<%BANNER%>

Using Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry to Study Modified Polype...

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20110318_AAAALE INGEST_TIME 2011-03-18T18:36:49Z PACKAGE UFE0012801_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 1954 DFID F20110318_AABSKY ORIGIN DEPOSITOR PATH woodling_k_Page_039.txt GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
9f4ae4c4bfd5c0192345892e867428f3
SHA-1
3f635a2994d925c48e294d28220b8d045606c8da
34543 F20110318_AABSMB woodling_k_Page_072.QC.jpg
7669c1b9a37e01d37a66576bdae9c60d
137310af88f207366bbd24efb31a821ed304f137
51813 F20110318_AABSLM woodling_k_Page_045.pro
c5b21868ae83090ea30c5cb3751738e0
6c859b3ba473e0fbc2ec2cb269f19c107ed26ff4
33474 F20110318_AABSKZ woodling_k_Page_035.QC.jpg
2e17b4103f59e750ef09ffa05bfc2918
a0a361f10d565e05e5f5c9f0ee23112cb59db9dc
898105 F20110318_AABSMC woodling_k_Page_053.jp2
cae54173c74716427d5a37a2c2d5eebd
1f73a51bf3a2e687b7a407be07a699e45ad29bfc
15158 F20110318_AABSLN woodling_k_Page_146.QC.jpg
a3ee6491d54e817e69eb870bc8f9e4e4
96988ca67092fad2f3facbb6e4a1d710c02b99d6
81794 F20110318_AABSMD woodling_k_Page_135.jp2
491d721ad9d5c401b24186404363317c
1ed3afba1dc83972e5c43825148bc007c0698380
1864 F20110318_AABSLO woodling_k_Page_059.txt
8e85cda664c1de41b1636d6459c9c280
7da07fd0d09455036bb473741b86992d9ebbf0c6
1875 F20110318_AABSME woodling_k_Page_112.txt
4f323f9116abf2e8735f0029629ddc1f
77e2e5dc8a034a126d0447c38f2efeca455a4690
779442 F20110318_AABSLP woodling_k_Page_099.jp2
a0972d5fbb00a76d7a71265c8fbf0c6a
7eae002a55ed0f9a225ab43170dd009d0da58179
1923 F20110318_AABSMF woodling_k_Page_047.txt
dafb33ed78dcf9d2bc0c1574299a1b21
8f0a2c1ed27fce798d95351ed60240f2fbb38ef1
880027 F20110318_AABSLQ woodling_k_Page_095.jp2
6c3f92bc0345edf6ffa3062109f78299
4b7e22485394969744a3bb1ad8905235f61b6ddc
8447 F20110318_AABSMG woodling_k_Page_045thm.jpg
748511355652c4fe03e47933944d22d7
bf17f2a4c07b1139be12046884310e6c70387f81
46359 F20110318_AABSLR woodling_k_Page_131.pro
6f351dbc1122a6da18b5b3da3b7c5dca
b535022002a4dfd467eaee27199b3dd8e2ad57d0
50445 F20110318_AABSMH woodling_k_Page_032.pro
07a79fdc97f54950a2a3c649e9e057d4
e80c83b9231707e07a8a6d9a21751d891c74674d
51544 F20110318_AABSLS woodling_k_Page_037.pro
c083fb7b3c23581dbccbe6888b38f897
3999da9d03e942c27a99293b8bb3eb1c4f9fa25c
6477 F20110318_AABSMI woodling_k_Page_074thm.jpg
206ef98148232149db51cc9cb232ab99
60fdb7fe1c8d21d1dbd95e8f0ace49f095f33670
1141 F20110318_AABSLT woodling_k_Page_005thm.jpg
939f3b00c52fa9be45f8ad6e9e612725
12e21a2bc0614595df1f22637f81a171f5304d33
81467 F20110318_AABSMJ woodling_k_Page_110.jpg
c123fda70b9c608b9fbae0bf7051de69
8c2e58bb2ce9174a0a7d6a5c216312473f916711
32678 F20110318_AABSLU woodling_k_Page_137.QC.jpg
43df2d285718dbec3dbac519186f069f
dd23d3acf1d6a58392275a6d76e5819611af402c
1912 F20110318_AABSLV woodling_k_Page_073.txt
8cad8b7d09f9fc5d2c4ff92a62c02971
c354454493bc99acb2cbafe69c9e139189509309
42926 F20110318_AABSMK woodling_k_Page_124.pro
e45d2f7722942abacae6c64ca3f5ceb1
8a2544d39e2913f3e7b38becb45809af7fff407f
105106 F20110318_AABSLW woodling_k_Page_037.jpg
ba3c3d00626430a24a1d86b54dcff0ed
ac4f2c8083fa0e18bcd91cde5acc1cb0cca72477
25271604 F20110318_AABSNA woodling_k_Page_066.tif
4b5c5c0b898e148d27f9d859814688d2
3faa41a5244c7cbfd3be708877de8aae025f1b73
5369 F20110318_AABSML woodling_k_Page_009.QC.jpg
8ddf1839df05532a9175f67401dab7ca
8c9eb1b3e6c05d6190c5d1352a25ab68e5abe67c
8379 F20110318_AABSLX woodling_k_Page_062thm.jpg
64eb04977e38ba10646e1179c8493e22
dc57036f1d073c7ec30ac899beafd11073851ec7
6648 F20110318_AABSNB woodling_k_Page_077thm.jpg
be639b5d9b0c69b37077a0038cf4bf06
dd75a3a616e581a952804bd1e42a3d574683e930
34398 F20110318_AABSMM woodling_k_Page_142.QC.jpg
1d7094bbbeb9cd5697901b30897abbf9
9fcd657740ce5b4519c8de300717f4ab14c623e5
8886 F20110318_AABSLY woodling_k_Page_144thm.jpg
de370cbc6be3812e4ccb073e72359c2e
c6bfe77886d980c428cb536f934da3efb899ee4b
44712 F20110318_AABSNC woodling_k_Page_064.pro
949f3c7add241e38cfebdbf862d325b0
27ec67bbefcb83c3b67e05f89e112e7a14add875
1968 F20110318_AABSMN woodling_k_Page_060.txt
67b66cf29bf5fd182c593a44224e2ff2
52378cc916cb3af59e60854723649629a93f163d
97036 F20110318_AABSLZ woodling_k_Page_073.jpg
be061ce63d93a4e527f81e12cb41c8fd
2c065cde866e563943a0990cbcb2450ef31ef0dc
859470 F20110318_AABSND woodling_k_Page_096.jp2
1f0aa20119903bba0e6651790199b88a
5635295ec8513f962ece638f3d7cabc9818ad295
46411 F20110318_AABSMO woodling_k_Page_140.pro
35e1e9bcf6b2e6a2123bb4995413453b
5a990a400435ec4b826292760ebb2cc89b427f4e
46756 F20110318_AABSNE woodling_k_Page_043.pro
c8bab9eb5486c973c1592e3790e849ea
a4417ebafedadb59e35b8d605ce99538847ba066
F20110318_AABSMP woodling_k_Page_012.tif
97bf38de689ab55c8cd2c46ad41028c7
717f7906c3c57e71f1d25cea1cb8763aa87026ba
52203 F20110318_AABSNF woodling_k_Page_020.pro
c20572cc6b862ddb285c3c4bbd8f3c26
f7af7a9f72926d213ea3daff05c202d27526b658
1928 F20110318_AABSMQ woodling_k_Page_025.txt
49026759b9bd1e0bb71b3e5b8d53f9b7
8b3d30b1572f500e26c0b487cf10586283c178d3
73186 F20110318_AABSNG woodling_k_Page_092.jpg
4216813d64f1673d3894caee2ce57a18
cfec33d42459d5ee13cb063b9e6fd0719640f183
108709 F20110318_AABSMR woodling_k_Page_044.jpg
d2f2ea6470aa2e46e7a229e9a0802064
d6a254214e69a14a35ff62acb1c143d5a0e58ce9
104643 F20110318_AABSNH woodling_k_Page_113.jp2
00305a4d5bff371b22ac50371c481749
65f05b78c6a003f4dffc422e1075fdf992eeae95
2557 F20110318_AABSMS woodling_k_Page_012.txt
7fe73619b6b22cf38c72318e86099b73
d3077ad71f15c6542a2b9dfb05201e518186acb1
47180 F20110318_AABSNI woodling_k_Page_013.jpg
fbf871f2f2658ddc86cab923b4214533
f8fe90d43743ccf0a7aabe3122c94aecb16e6a5b
1170 F20110318_AABSMT woodling_k_Page_003.QC.jpg
58f522c05c252188c6cd2ca6c53da936
71b1a7e14f1ebaf53a15e132ba35a833f07274cc
8836 F20110318_AABSNJ woodling_k_Page_101thm.jpg
53de1f555eb3a81a92d88f2f71920f82
49a526e5e0107c55022b11cf8eea85df7c53dfd7
7133 F20110318_AABSMU woodling_k_Page_019thm.jpg
653dd1580e30881ec29976e94a283543
9a5b572f8117dc54614a864ad283c585ac1c4728
7308 F20110318_AABSNK woodling_k_Page_052thm.jpg
e1abc8e58b464556ea0aa64bbaa5924c
d31f2afdfe95f56679f43c17bfdccd202968c9ee
F20110318_AABSMV woodling_k_Page_090.tif
a54b0faf4e815babe759f1f939198f54
ddd5501750ca903baa1e44bbfecfa1f628d97c66
1973 F20110318_AABSMW woodling_k_Page_021.txt
d2dca84d35731c9fa55a66db28bfe7f4
eb6e04daf2811c3c8cd97cfc94a029ffcf6e891c
1711 F20110318_AABSNL woodling_k_Page_093.txt
cb9f3aa0df59b2c93611ba6f5c1eaf26
53d1a283163c00bc3656eed701c2d37424f02bef
1053954 F20110318_AABSMX woodling_k_Page_014.tif
4c2926339db4c08b1ceeede9dd2379e5
b6d992edc3972a9882f6da6c9adfe5fbcccecd72
29904 F20110318_AABSOA woodling_k_Page_090.QC.jpg
b06d5a618670a6100b8cee603575896d
abed8d602049e8ffe5d11da08ca895a05c0967a8
48698 F20110318_AABSNM woodling_k_Page_039.pro
3d78edf4d4ece3baa3c39893b9d05fe9
199a1a3c4f88f4a524bf17361042a209c194481d
46290 F20110318_AABSMY woodling_k_Page_146.jpg
97a5a0dae9943d7f87d7a1abe62fe758
a70e62083b25982b3490519b6a25dfcee431482c
81970 F20110318_AABSOB woodling_k_Page_097.jpg
1059c71461485418f80f0e2ae125b72d
a18ce95b4c3d98ceccf7b0511e2e4a9fb9bce987
43867 F20110318_AABSNN woodling_k_Page_022.pro
75cd38b59a74f30a08a106a683e395c6
6a7f8ccc22b5515bb8518ca89b2e1cea7f29196a
68549 F20110318_AABSMZ woodling_k_Page_130.jp2
3b4f331f538b8ca47b892e898a04ca0b
c0fd35e3c37a6e30fbdcd8f45070726b9df67e26
1051986 F20110318_AABSOC woodling_k_Page_006.jp2
1ccab9cfe5fadf1cb4f950ad1c544921
656fae62c91bab572a017739765287b84a534fdf
31375 F20110318_AABSNO woodling_k_Page_063.QC.jpg
65c5bf4823cdf087c84572268bcce6ea
f66e1517d5e7a999517f6ee44e90fdd30f0150fd
20522 F20110318_AABSOD woodling_k_Page_146.pro
61945cbffcee44b3aa16a2591bb4eace
a4237b8b36ba6a4038bacdefa8fdf4d50ab9f3b1
27943 F20110318_AABSNP woodling_k_Page_053.QC.jpg
1c9afe911fd54be387ec1022e0ae95b5
7bbbe982d93cf5c459e1e72172400c5be44a52a7
F20110318_AABSOE woodling_k_Page_032.tif
3f33efd33fd711c37e2915b62b16c4fc
03e213a9e58b49d4b3f2b71b286e436678d1b10d
156734 F20110318_AABSNQ woodling_k_Page_007.jpg
eac32db9a96b91dcf1b7de5189abe7d9
cc9850f64fd3248bd2148eb8d0c6e516ac59e316
95773 F20110318_AABSOF woodling_k_Page_136.jpg
7d35c749993c3ec92af59d08cff30c2a
7bf1ed65a1ffe0c951057f78a18fa4aca64ae1bf
7389 F20110318_AABSNR woodling_k_Page_075thm.jpg
65e4671fd689a84ec252836a1298889e
77d1922efefd4a2cfdd56fc5e252999b42058b8a
7381 F20110318_AABSOG woodling_k_Page_091thm.jpg
2343ad499fcf18379784ca0109a063b9
9fca490337a8d66629fe71cff25b7cef2be0cb64
40724 F20110318_AABSNS woodling_k_Page_040.pro
88fdf59de46942d8d91c7bb4be3ce035
cd39463d45f97b0223ec9060ab9afe64535d3372
95502 F20110318_AABSOH woodling_k_Page_117.jp2
20a0f982b5f2ae455cd3925c4221cb10
b1dc552cc19be570aa38206d408c0b8a53651ead
937901 F20110318_AABSNT woodling_k_Page_089.jp2
24acee970ccf672e7b2ce5fb75b42c3e
6b9210ee998f58b311415fa6c0d9377eaef0efcc
1639 F20110318_AABSOI woodling_k_Page_075.txt
a88283f32118e0ef826bebb5814a72e3
72b345cd768e28a03e4f073ac29113d1dd943e24
6208 F20110318_AABSNU woodling_k_Page_123thm.jpg
aacbf993e2eea9de4c16f46d443c6ece
c8750a3fdb61e5c4467d707e3390fcf83c62d7ca
49275 F20110318_AABSOJ woodling_k_Page_018.pro
835d7cdeff12dbbb46b8e9c7c45ddb95
81faf6a65e12d4c2c9d71531ccc7bd38f12a49f6
1003560 F20110318_AABSNV woodling_k_Page_124.jp2
f1243d714031ef95c7e57cea81c98314
4fee83c000d35592390bbc3f6a3d530815970099
83866 F20110318_AABSOK woodling_k_Page_075.jpg
8391ef117ae0bd10fbd5c4fab68f2367
06e6c039dff3477d6ec01a8de97284004aa619de
84686 F20110318_AABSNW woodling_k_Page_095.jpg
5afba15fe1c64a6cdcf1013fb54c366f
877ca39601b29a1cf0efe53102afa53cfee5e3a6
F20110318_AABSOL woodling_k_Page_076.tif
db559563a97f01b1fbdf65da0f446f91
b0d07dd9b0eb56101366d90149ca8b579df15747
34270 F20110318_AABSNX woodling_k_Page_143.QC.jpg
551b9846c743998e88635e3ba7147119
bc5cd575b2458742cd4082385bee592acdc3c4cf
24004 F20110318_AABSPA woodling_k_Page_080.QC.jpg
f613a83122dfdb81dad8a3c28ac02fa5
d7a152ec2d0476a34d09a164f0fb9ed65e567e36
F20110318_AABSNY woodling_k_Page_009.tif
9fd8ff02b4ca96b4971e3e567efb6e99
c27c7fe70fb41aa1173a74dd81685b88b7a6122c
F20110318_AABSPB woodling_k_Page_111.tif
d629fc7b103dbcaffbf2fc5d2c7ecf53
0929f3b75cadada6108f9b405ced3c0206b34592
28963 F20110318_AABSOM woodling_k_Page_059.QC.jpg
236de173157aa83f6162eeffbae24558
2888aaf44e2a4f85318a9eb39923469b9ee5a83a
43406 F20110318_AABSNZ woodling_k_Page_105.pro
d53362d6ebbf28c01c0b9beaa056b692
a751c0213a5928526d155bcb72680bed0d3ecd29
1763 F20110318_AABSPC woodling_k_Page_068.txt
4b4439a4a36d819a3a65b1420aff059b
004e37e5c54501c31522c86ea5c0766709be4b14
7722 F20110318_AABSON woodling_k_Page_055thm.jpg
ec67c32cfa61b982c7b76412c2b4df58
41b97eea53500dbdeb5eebf02478e1d9fcf827c3
F20110318_AABSPD woodling_k_Page_140.tif
8fb4b8e6d5c758c2b24c378f6ceb2521
6db1c0821c6ae7ba7bd03498b57b3beb815a7a6d
F20110318_AABSOO woodling_k_Page_027.tif
abfda7ed4b1ee62277ac9ea431ae3a3e
3174193a00ba4eb44bf3d189523f0853802197de
98638 F20110318_AABSPE woodling_k_Page_091.jp2
4055320c366737d64d03d181f343eb03
9ee41f73968eead4564a5cca5361520ec063debd
F20110318_AABSOP woodling_k_Page_049.tif
16b0cd8e220ca7449ac6741dbb142853
bbd763c57aa98eb9e9274f2c18035c7a205b9e33
F20110318_AABSPF woodling_k_Page_077.tif
9bf64cb29caa3e5a73432a280476e189
0cf92a44bdb4e1c4e9983dd3ed1585c4ada85736
34995 F20110318_AABSOQ woodling_k_Page_087.QC.jpg
e8b682105d4d4dfb305bd50bb3534729
83659f482b5c99c3c792ce934073bc162f69273a
44393 F20110318_AABSPG woodling_k_Page_138.pro
795202be3a099accc60c6ab20281dd0f
9b6ff260d6c598ad07f6d30ade0fa721847d9cb0
F20110318_AABSOR woodling_k_Page_139.tif
95ad240b753093f67cf202b4c5945c30
73845365629eadbbf8b638d79d523bfd31d10630
2022 F20110318_AABSPH woodling_k_Page_037.txt
eea5c307f009cd41135c46de2765014b
de183b6ffe2013ac6e0a55993f2a004321fff32c
91385 F20110318_AABSOS woodling_k_Page_042.jp2
814d51344e7526015e3fa91d1325b8ba
51cfeec24a8a67a7842fa1feaeca865ae9331009
8486 F20110318_AABSPI woodling_k_Page_036thm.jpg
ca4d4b5dddef853c445e71b210469f0d
1cf238c8aff65d30617f36b16251a48b2c3f2992
F20110318_AABSOT woodling_k_Page_084.tif
7ca993285345e5a6b792c3c81c667d36
b287c796dac34c380e45d19ff01a177a8c4d41b3
17806 F20110318_AABSPJ woodling_k_Page_008.jpg
ac8d35d2bb0243cf737f4721e63a2291
76d148323d93745f72beb84090209bc7f234a630
F20110318_AABSOU woodling_k_Page_025.tif
4ab4364e81833ac0b59b4c664ec0d522
6c11e55ef5dbe9a459c671753a8b50093b5cd0d0
8215 F20110318_AABSPK woodling_k_Page_069thm.jpg
08359c27e3d8a3439282ff13205c682e
7fceaa3641334428ddd25ca530f1eb559cd8afbf
23923 F20110318_AABSOV woodling_k_Page_099.QC.jpg
f2e87068f570e85331c3f13a02241e0a
612428c8f12b8da3ec6ed9a8d7d4f598aa79a8b3
8085 F20110318_AABSPL woodling_k_Page_119thm.jpg
2598ea44af744089491719791ea661b3
56cd9e497441590d50c1bf7f465fb890f9a1f562
21948 F20110318_AABSOW woodling_k_Page_082.QC.jpg
289902a6bda7fbfa1536536d476f244b
5fa285c88f519ac65b10a4b9fcc171c8149f29cb
48782 F20110318_AABSQA woodling_k_Page_054.pro
01da159384c2c87f417f42002db77984
8d0b52213c8274167dae551c8743e67d25e0a448
91799 F20110318_AABSPM woodling_k_Page_103.jpg
e767f1428db5313c7df35cd435bc2448
308475c156ae5cd5832dd86f98e3aea9e300a1e9
F20110318_AABSOX woodling_k_Page_034.tif
52ea188a0dbbe110467b326764349a84
218e948ad803380f9f43934cb27b6be92bf040ae
104376 F20110318_AABSQB woodling_k_Page_047.jp2
eafd5dc7364dab1d0fe0864b7ef87948
9f5fc5c39e8faef0a3593c1e407a54b05a0508a8
26643 F20110318_AABSOY woodling_k_Page_110.QC.jpg
827ca53d80882de4145f234fab28b053
dc635d38209888aea34b00984b750623a42a66c1
254726 F20110318_AABSPN woodling_k_Page_009.jp2
f4c7e58308145c053ab07e6a1d4d9f77
99c4aa4934f0ecdb57de29b19d24df102fe97345
33782 F20110318_AABSOZ woodling_k_Page_031.QC.jpg
7770fecbd27fab3600bf18e63ebc7879
f8a55d7016b668b4c2def1ab92b18fb77094e2bb
104528 F20110318_AABSQC woodling_k_Page_026.jp2
3c57a913af5c4d9140a3dfa9674adfca
4cb20a47641aa24a4ea3582d384935bcb2156e34
107328 F20110318_AABSPO woodling_k_Page_134.jp2
df43e2fbfb2ce04821673f991f562d24
d3b3a9df3bfa67f051c2b3595452ee685a6dcecc
6035 F20110318_AABSQD woodling_k_Page_041thm.jpg
d9c676b7e500078eb1a7cdf5b62bc20b
d5a1dabb697bd17025b33ee752ba55b4c67c8eff
106211 F20110318_AABSPP woodling_k_Page_039.jp2
bc4ce5219c681b1a16ad31aa495f5f6a
f552421e05163174c83e27e9dbb5eb7db57b588a
109275 F20110318_AABSQE woodling_k_Page_072.jp2
edcb4607956358405d737fe01a1582ac
678f842be61b89e9a5c3cea998d17956ff54d883
32245 F20110318_AABSPQ woodling_k_Page_073.QC.jpg
9a24b0632bee5034f40c9b45e9d13556
c1d362c9338bff842cd7381207bbe5e4e59df5f2
F20110318_AABSQF woodling_k_Page_006.tif
31166ef379e0248b13257ce9557db6ff
234d00172fe6989c66d5ec3a1ff7d44263880ced
F20110318_AABSPR woodling_k_Page_141.tif
67a1bd018bff841782cf003b8f042503
252bc8a781fc402263315e740f40b6ada113ffcf
F20110318_AABSQG woodling_k_Page_131.tif
f53aced4876f69dc0b69b03acf7844d5
482aa924899fa8608b3e4dd4c4ea266190c09415
34942 F20110318_AABSPS woodling_k_Page_062.QC.jpg
69c8cf435ca403cedcfba8a3d711213c
a1e259d96cd8569a9c2841c2cd7a3f252a76fdb2
1749 F20110318_AABSQH woodling_k_Page_091.txt
6ed1907c25f5b5cb1636d0cf8ae7cea8
a7b13f471525cb911c517d1f1dac8991519cf766
101711 F20110318_AABSPT woodling_k_Page_115.jpg
f0726cbc8a26317f048ffa8de628b4cb
e511e93a141b23623b2df85527ce21d2533e744b
139735 F20110318_AABSQI woodling_k_Page_012.jpg
0a9394848e3331b7ece909f192ffa8e6
8e890d863877ad4cb291f0731a10033b40caf289
44637 F20110318_AABSPU woodling_k_Page_059.pro
47d520fbb971ac63c39d9eac780da4a0
758ae64d362d9f3d98ab6190b3596f0c7c90506a
28158 F20110318_AABSQJ woodling_k_Page_056.QC.jpg
1a92e03fce12f27203c028d449d02006
ca128cb53ef5733a35dedf5bfb329fe0d601430a
59432 F20110318_AABSPV woodling_k_Page_010.pro
f0627cff60bb765922211b1258995fed
7c5f891501ddb995c3445bbabc39fed136cbe731
33940 F20110318_AABSQK woodling_k_Page_058.pro
ce1f4fcd97d4f45e8ca625c845af2ef9
29be386a4099baf91a003bbaca0f77b77a0b1d46
7131 F20110318_AABSPW woodling_k_Page_048thm.jpg
5eb4b4c5a34dcb4cf5d007a1f913c6ae
6195bd962aa23d7857dbf18f65c78ef3df96355c
801331 F20110318_AABSQL woodling_k_Page_050.jp2
01c6233357d01ee6df0b6ec30dcfee5c
4d9fade9ef86fc4edf3d0dae602434b642acbec3
2089 F20110318_AABSPX woodling_k_Page_022.txt
6c82bfa8d600a6ed8753db17fcd75e50
4f3858d65984acac45fc39380b937d8e2cd80efd
1051915 F20110318_AABSRA woodling_k_Page_128.jp2
c15f8c05647017a070818575d7ca4828
5289131e0933e229f79af12ddae7675684c5cb24
8647 F20110318_AABSQM woodling_k_Page_108thm.jpg
e39a80659fcaa8f8d6bb85a8face44ff
7438b4d92d44c4a715bd46df33ca09fb038c2d5e
32386 F20110318_AABSPY woodling_k_Page_034.pro
45e98e4b407828a6b81a498e3f959666
66075b6ffa5120f0033e5a89ff44cf840f1e6ddf
19347 F20110318_AABSRB woodling_k_Page_013.pro
3e8106b1460e2bbff6b3f539c18943c3
b7d5549e5fc9b5468acd0a54d75c1bdc2a850b51
34693 F20110318_AABSQN woodling_k_Page_083.pro
e9afab1baeb0ae33a2bf79b80649bf67
a16fec19d6b43ff96acf9b6b5d112964c43e0d97
F20110318_AABSPZ woodling_k_Page_072.tif
6ef440b6b6a620ac567b93d933fda1d2
5b790c1d4bf799a79a809157573b8791a9e40f39
7291 F20110318_AABSRC woodling_k_Page_117thm.jpg
340e35296c58565df1584d88b06e5417
8af1e88e20deb57b4e3dfcb75154703d0e02dfc4
22464 F20110318_AABSRD woodling_k_Page_092.QC.jpg
dfddaa6b5bb9b4dd98c223f482e63e88
37de73cfa586825c3fae5b66b9a376ddff891a1e
24238 F20110318_AABSQO woodling_k_Page_121.QC.jpg
29afc5206d590243f7b3394309acdd5f
f64e5d6db2205ba67e4002d3f848c4ee13b70a37
F20110318_AABSRE woodling_k_Page_114.tif
8b247f1d7c5000df7834cdca5d929d3f
7be0d87b05e9466858aa1a6354adce76154b618f
92075 F20110318_AABSQP woodling_k_Page_065.jpg
df7df0b0440b74b6ba214f6beb14804d
71372b8f04f395134c78dc91c43a5bd65ea77ee8
78609 F20110318_AABSRF woodling_k_Page_129.jpg
1e2531bd7510a1a2e36e1103030e12f8
fd2dedbb687faaf0a4babd092075480cac208be8
1051953 F20110318_AABSQQ woodling_k_Page_144.jp2
5bca858f502635728432cd4e326660f9
d8197727d9fbe443ab2609fcc07a98b5032c06da
32809 F20110318_AABSRG woodling_k_Page_119.QC.jpg
96150f27cdcd63f22f25f7c77e49b23d
a4945d8fbbbc531e722564acb9ec51c3adc777f6
8482 F20110318_AABSQR woodling_k_Page_133thm.jpg
551b87d0f70b6020880608222c5b71ac
d4bc136da93f5d2bd48dd4793c379be0f6fd6c67
8214 F20110318_AABSRH woodling_k_Page_007thm.jpg
d7a9010b094da8af1d7298ab2411cf1d
908043d35f80e820db8b373d863a75a994641163
93387 F20110318_AABSQS woodling_k_Page_056.jp2
1caa889c26829096baf9792bf497bad9
10ef1adfbc9063f57bbf6ccc049830c46e1703f4
838100 F20110318_AABSRI woodling_k_Page_038.jp2
495d8c07a63df6269b808ca5a7d76742
ae12f835981753e5d348a56efe9bb208177b96dd
1051851 F20110318_AABSQT woodling_k_Page_101.jp2
2f7825eb7c6ca3d4ac1c6cfcec6791df
272395b95d7e8009c28d1180288e021a32f1497a
103107 F20110318_AABSRJ woodling_k_Page_043.jp2
935166ea442867fda6354370d3242a36
3f077fe375147eb092129570b377ee108e0c29bf
30294 F20110318_AABSQU woodling_k_Page_114.QC.jpg
110cac21aa51f0b14dd0c6aee75c53b1
9b7430b9423ca487092cc26d7b000f8ef646a360
713398 F20110318_AABSRK woodling_k_Page_082.jp2
a49a811263b28071f55c9af4e0d71b8b
88e8cd9099b99a308345a2dc4e2fd2586e83d0b5
25919 F20110318_AABSQV woodling_k_Page_050.QC.jpg
4d9aaf7b0a2b48c93a750d4afe60f899
2494f14e95eb3841e042f5fa541fda3eb3fad665
31836 F20110318_AABSRL woodling_k_Page_025.QC.jpg
75fbcd660e44b4d2f6f2db1d222ff3b1
52e826c913e4f9c6d1d82f4e1f98dce92fa5ccb2
1051978 F20110318_AABSQW woodling_k_Page_007.jp2
07b6ad1b7fba2410cbc490149a9d402c
2251c64dea30e2674eaf7de70a8aa02a6b663780
7000 F20110318_AABSSA woodling_k_Page_097thm.jpg
5dc45cb2a491e51c08d1e9d6659948fb
fc21a1036f1d2ea55a53aec849ff3904ea3f1553
96528 F20110318_AABSRM woodling_k_Page_139.jpg
33e4a600a43c6590f58c4eb36d899fe1
d33bb1c033721acbf1eef130d2cfe0140c202ff6
37487 F20110318_AABSQX woodling_k_Page_145.pro
25728fea34e76d73ff5f27affc4f2d55
f0bbb65504bd32125d908f2de30088fe768159f4
85825 F20110318_AABSSB woodling_k_Page_016.jpg
18efae132243e3b383db5390fce21456
a9c5610dde1c9947b88500422a56a0eef9364a42
48933 F20110318_AABSRN woodling_k_Page_133.pro
564f868ef9579a0217f75c506e4d4d1b
d19345240ee2192b7f7d4015a9825585b86690b2
65178 F20110318_AABSQY woodling_k_Page_012.pro
91d4d9aafbe0fcdaec6a51df946c79a0
d3dda342a35c14386dfd4c374bc438a0ec2077db
867429 F20110318_AABSSC woodling_k_Page_097.jp2
15acb91ee90e76d35f1ce987ac1a2f8e
730921839d447c982532f30b269c27b83c50c49c
1635 F20110318_AABSRO woodling_k_Page_129.txt
a1fb7caa3d0e9a7d061dead88581b35c
027a267b71949c26fd474441a0615215060880e2
6580 F20110318_AABSQZ woodling_k_Page_049thm.jpg
bda3987ab923ac248276d48576053e9e
b5445bfabfaa7b458624c7b1edd52da2768b7948
34301 F20110318_AABSSD woodling_k_Page_020.QC.jpg
a30806421c7690ecaecc8cf518d05df6
a64aadbd077a9ad218949bb3763f85126662e2f7
F20110318_AABSSE woodling_k_Page_094.tif
afea77543dba6a438aedffeabc87b8ac
7a8eee63ede36f82822351878693be51ccdc2616
8464 F20110318_AABSRP woodling_k_Page_023thm.jpg
67920b58c612a87f1a476d405561dd38
7f668733562e7df71786cf4dc3a014275599f929
1702 F20110318_AABSSF woodling_k_Page_077.txt
936a1a834df1a5c34b84ce7927b72207
471382b12f9c58f552d0f873c5431d4ad97f21b8
868287 F20110318_AABSRQ woodling_k_Page_098.jp2
54adc52a62a8c34d787a4219dee2077f
3ce6fce6028a00b1e767d558aa97bc301004043e
25676 F20110318_AABSSG woodling_k_Page_006.QC.jpg
efe102645a7b32cafa2aaaccda197961
65855f9d29206f68907cf14e4e670c0588dad499
F20110318_AABSRR woodling_k_Page_101.tif
d4279673822d0b3b06a9eb57a360a87c
e3db6b4e2d24fd9ba780420638b1c06c1a7f564f
24435 F20110318_AABSSH woodling_k_Page_058.QC.jpg
6fef1bb5cd77c6512f1e100920f7c7a6
417f16fa726e7c62bfab85f1a425edc7279f658e
996833 F20110318_AABSRS woodling_k_Page_104.jp2
45d13358ea4e30b24fb585d34b36762e
c21156b15424c25ac655deec1cf84f6929286017
105888 F20110318_AABSSI woodling_k_Page_023.jpg
5161c5acfdc1b30b9542e192e3d22ca6
41acef0c9cb6b8259567fbd3d175d7a13ea7d2c1
71202 F20110318_AABSRT woodling_k_Page_074.jpg
5f155aa2cf384adba83602231afd848a
4641be27a9d659f0469f1f186c7325c8ed6d405d
1587 F20110318_AABSSJ woodling_k_Page_080.txt
ec1b47fea3b30eec5ea86369bcf79590
6d0393f9c86bb1c0fcf6bf9b31146b407d7ec0b9
91708 F20110318_AABSRU woodling_k_Page_064.jpg
592e3f6239dc90f752e0e98208779290
3c45441303ef262f13f29ad7757347f00c9231fc
42364 F20110318_AABSSK woodling_k_Page_019.pro
f07ae853d02e938abadb941b64c0545f
7d00dfd7ad44969a1ab1af31fdf673cdb5f402b6
F20110318_AABSRV woodling_k_Page_109.tif
76b5a27f3cbb693ffe36e3f05f505dd0
9b5aa1aeab08f7c91ed20aa7ee75207de86763b5
8535 F20110318_AABSSL woodling_k_Page_037thm.jpg
1da0c585b57c2e99b648bd7486134a1a
62ab9ce99762a0d38a11121fa7c269ec73d8e01e
F20110318_AABSRW woodling_k_Page_146.tif
b1ab1d6518c59ee0dfc4d2a79ee60234
f0d528c19530079e7bad6c5f634c37e2472cc4b5
48851 F20110318_AABSSM woodling_k_Page_025.pro
a805098a4c2225c46eef26cdd7cc9d95
c016e4e131cec61d34bf54fcff23c3dc5e62b1b9
104384 F20110318_AABSRX woodling_k_Page_073.jp2
6e710bd8c15d6941bfb174061f918b45
57493f309c0bb5cc97faeb364cd5321f62ba88d5
69409 F20110318_AABSTA woodling_k_Page_015.jpg
9e3e4a91381c046600082e06a73b1d62
e8cc847ee50b10000a3ce4ebe0911da7c24e9083
36475 F20110318_AABSSN woodling_k_Page_049.pro
644239f84b133a811cb4431f008058cf
7aa04c9939516a811859dd754cce09c969bea0e8
65045 F20110318_AABSRY woodling_k_Page_011.pro
b23447241ee337585655cdd9d8f6035d
b7ec9c9ed049a0f014fded037767a1bb44203071
84452 F20110318_AABSTB woodling_k_Page_093.jp2
d5af21b80f19f41cf046b979bf8b2aa9
da281533a83a6928c97964a9de870da00bf6c067
113186 F20110318_AABSSO woodling_k_Page_027.jp2
d3047296518090b886142d95cd7ffed8
2c705e33c1c9c691f393e8f3f345d32f41ec2eec
1051962 F20110318_AABSRZ woodling_k_Page_010.jp2
2dd5c99cfe58cbe1312be7eb0da598b9
9d87d9572d9f795dd239c5c578bbc15613db97ba
3239 F20110318_AABRQA woodling_k_Page_006.txt
bf12e9954fffadad236ec31116432fa0
4f7aa16afc1b3af20d2105c8d24a441a65b33f26
F20110318_AABSTC woodling_k_Page_133.txt
48b424ea53a4e0ce3944f7287f412743
26468681da085724bc8c19a7973f2fd715b87d31
2110 F20110318_AABSSP woodling_k_Page_027.txt
4be85c63f7248870a5b33eec0a394c33
ec270b2501558b66753abc8813fa648086d8c919
753142 F20110318_AABRQB woodling_k_Page_074.jp2
d773356e609137aec9bfbd43bc4ce21f
76b62cdc44a241767087dccddfaf215cd9f6dedb
7174 F20110318_AABSTD woodling_k_Page_122thm.jpg
05eee5dc5c49b18c7d960365af634402
ad031c424568598d6cf7b115aa820ab45b979605
81352 F20110318_AABRQC woodling_k_Page_145.jpg
055ac708ac5e1f319011e5fc33bee41c
b9bd3c8dcf78747b3dd818d9338762cb256f3f6a
69329 F20110318_AABSTE woodling_k_Page_120.jpg
474a46b1225195ea32bdd2281716c815
b2fd48d24dc9d3d02d7322ce1c8ff6135017e664
F20110318_AABSSQ woodling_k_Page_064.tif
16b87bbdecaa6e776f0d751b60e6e322
9d4bfc1b127c9cdaeb79c0e1b3805428e94dcb99
109927 F20110318_AABRQD woodling_k_Page_031.jp2
9c54877090d1faa12ae5e7c6af6a2607
915afbac99920a19a9dcb6cdcc2291e3a8b28947
33222 F20110318_AABSTF woodling_k_Page_015.pro
76a51b063ec48907a7ca3ca426056c19
c1d65fa8e7a4cf51ecd91b6fd23b3b33ee4456f3
1430 F20110318_AABSSR woodling_k_Page_099.txt
2d20fcff5a0d46b702c9ff6e96c4a9ae
9c55b05eb3103235223419a22989f26ea6025ebb
93491 F20110318_AABRQE woodling_k_Page_055.jpg
6cba85a21fe58c08a3632dbcb3833955
43e4988abd840011a1bab348c6494e8a3f846eeb
33747 F20110318_AABSTG woodling_k_Page_088.QC.jpg
52b6793fa673d210706b8ea705677d56
ea640c8394baab75936d7769f505df4e0736605b
1733 F20110318_AABSSS woodling_k_Page_002.QC.jpg
30fe321613f9271fac2477ca353ba884
6ea28b22301d24f6dc0dba7c4f9374ec988d8194
11468 F20110318_AABRQF woodling_k_Page_005.jpg
e2f7b487023c44a3a57283276b8e6a9e
b1d9bda03f86ab4901da2f027ae85f446a479f9d
34641 F20110318_AABSTH woodling_k_Page_041.pro
7c3dc3391cd56c79e84ea227d9be569b
b2e65cee9f5d7c0439f37cd90baa54866c376fb0
30195 F20110318_AABRPQ woodling_k_Page_091.QC.jpg
620c3b290948a12c65f1296217de268a
6dbc2f29d233374ea068c557553d509fce1fc527
39315 F20110318_AABSST woodling_k_Page_071.pro
ba3ea29fb49220d6d2dc7c846873a042
36167f28793de5bd5c6cfe59e6d590f5a843392b
32940 F20110318_AABRQG woodling_k_Page_110.pro
bb0a1a90b365f19de63949623e61c5e7
014eeb514c3e5bd2c4b4ff8f5f1a54e025cef77f
29269 F20110318_AABSTI woodling_k_Page_104.QC.jpg
5842dedbc82364b30c1f2a6d81e56974
36a77484e1c6c6d1d6ca8edc10f278e9934e7a8c
97112 F20110318_AABRPR woodling_k_Page_078.jpg
7a8a2bf51d60318743c9888a721c20af
6a6ab78371dc948020e39c7e3f708de8703b84e2
1421 F20110318_AABSSU woodling_k_Page_083.txt
6ed9def6aef99bb799c2a17f8a673615
b6ee84d9925bc88c9c75ea6540c4ebc111cd116b
1443 F20110318_AABRQH woodling_k_Page_061.txt
19468fcafc06ef2d4d2c9de481409cf3
8cf3bf75fbdf8d5784df78928027b5904e087a71
106331 F20110318_AABSTJ woodling_k_Page_027.jpg
465c7a569eaa92460ab8f9c4b5b24502
fd6b96baa65e6a23a3161b5a6ce5acbbef8966cb
80421 F20110318_AABRPS woodling_k_Page_052.jpg
3ce57a74fee914496683577477a7a20b
2248ad31f3b7436e6ce2bcbad697b4c8f0e4d5b6
34901 F20110318_AABSSV woodling_k_Page_074.pro
deb43a67de63ec8f3cd9c4192c1b75c3
0f92ee6b97ae90c392c9475465eb8f96c75c5780
94422 F20110318_AABRQI woodling_k_Page_138.jpg
82d8fc21360dfac318def3648211dea4
3038f78153aa479d5d687ea5cf45a461abf9e3b2
98791 F20110318_AABSTK woodling_k_Page_139.jp2
c1df2ee1fb0fc185315af893bbe06d62
6dbda6f2da375a13531068efad24eebe63f50e15
736890 F20110318_AABRPT woodling_k_Page_079.jp2
08b42b739f4f6feadfb70d1fde3f33b9
65fb58290d4f5aa3729263b4f847ef4cb906aa16
30183 F20110318_AABSSW woodling_k_Page_109.QC.jpg
bb48ff4b00feeb42c8ed785cffca41f7
b21c6f0df5f5f66774d2e13a4457db23df10df36
104281 F20110318_AABRQJ woodling_k_Page_025.jp2
4ea6d3b1f182d30eb8d4aa9e787ccc0d
aea9025484a40328a400a9c24d892e996593a54e
F20110318_AABSTL woodling_k_Page_067.tif
9c1eaf17c35ff724ae4224d075218d83
9f87c94dac391457a26fdc05d7a5a7c147437c80
112870 F20110318_AABRPU woodling_k_Page_143.jp2
11ffdd802768258766c59e29f51b7d44
2377b1496d12b1057efdcae4101edb6aa0309f71
F20110318_AABSUA woodling_k_Page_128.tif
6a635f63d84460352d14162937967259
95ca9ff6ab700fcf49044d6cc292e2b5c55da102
33052 F20110318_AABSSX woodling_k_Page_123.pro
ee49299356014db4b588b6cd955d7502
7a9a15659a4212153c828e8d550eafbb2484b7a4
78897 F20110318_AABRQK woodling_k_Page_038.jpg
6672c7107186661a2426788eeb1ae6e7
bf238e6bb2251b5a635c65fb8105998cfaa3cec6
1863 F20110318_AABSTM woodling_k_Page_124.txt
78715f9f1cd57a2b2920bbc2b86ae071
e673cfb3367c2109b93fe874ea6fa4cb5f00a296
F20110318_AABRPV woodling_k_Page_008.tif
6564928100ba07e8df9e4433d9f7b0c0
6b182c5d4db68b7adb8a2a6ba1c8f88b6d1069e9
1528 F20110318_AABSUB woodling_k_Page_053.txt
0e1f70cdbac58bd3e31b22f39e87423c
b03561eac568cb709514542d3ee9ee12d25a19f8
49288 F20110318_AABSSY woodling_k_Page_017.pro
355c3d20d86361e8fc85a5192b6ac742
a249d0e0a1ccba159edd48ef626d9438ffe999f8
1939 F20110318_AABRQL woodling_k_Page_118.txt
0a85bf087f61b25f9fea4861ed43c173
6bca358732242875b8a36a7e4b4a6aacde19ef2a
2592 F20110318_AABSTN woodling_k_Page_001thm.jpg
9ce0740451f74be370c97e6fdba61bab
4566be22ecba006d91e5850796f161235e74fd2c
80277 F20110318_AABRPW woodling_k_Page_122.jpg
5b4d3f22ed37ae54c480f3e7953d35dc
e8191d0f7a1b7a5b1dea3dc6f8de6eebd86da5dc
30697 F20110318_AABSUC woodling_k_Page_055.QC.jpg
8453f030efbfb8e0ed44d912adf39000
8f44661d30bfa36bb9a4f5a994053aec117450d1
107663 F20110318_AABSSZ woodling_k_Page_142.jp2
7804489634aa56a1e49531d6f9b2fabc
64beef9f21a418e33da1fe48b376e0340fec9928
100028 F20110318_AABRRA woodling_k_Page_131.jp2
470f88a8d0461739287aac6ffb12f8dd
53422ed73561ca236c4c43207d379b76ca559a57
7325 F20110318_AABRQM woodling_k_Page_084thm.jpg
ddae67b7eff22ba57731333f10a8697f
2cf3e4f75431764a1712f9b4c142eb6b68b50e70
102667 F20110318_AABSTO woodling_k_Page_021.jpg
608efd09d53b5f384a4aa928b0cbe9a1
f678cb926db3cf57f9a0eee134fe8e5f428a5508
39917 F20110318_AABRPX woodling_k_Page_014.pro
baed2bd847423bd53833b24661dbce45
0cdd5dffbc585db1b8030d845031a36085f9b7d8
92 F20110318_AABSUD woodling_k_Page_003.txt
4e9f51916601e9729925fa638320a1c0
4e67df3c01f004e90be3454df5022bac6b3018de
35467 F20110318_AABRRB woodling_k_Page_011.QC.jpg
0edbe0cba9964e1e5e09f6a7b8556bba
725bbac5439ff8b58179f8f8b4e52e26708bca22
7086 F20110318_AABRQN woodling_k_Page_038thm.jpg
a706c7578d432142dfd803f184f84332
9054b4537e53fdf07c825394c40a524f5930b4b3
22085 F20110318_AABSTP woodling_k_Page_120.QC.jpg
33f1a6df3412ddb000983eaebc6f7d3b
22c18402b9d64340694275655a7fd06bc9dbf530
2017 F20110318_AABRPY woodling_k_Page_038.txt
fcd410b394f3839ed8b6517be09f24eb
3178f768c2aa521a6a2979a2b2b6e4bfac885ef5
72612 F20110318_AABSUE woodling_k_Page_079.jpg
f93d37ef32b4bca42759b748b8d4c949
e63ff8730ecb336a0c948981d47739878cb27860
110616 F20110318_AABRRC woodling_k_Page_062.jp2
df8f4ff72415329e629b6fbf37b0d436
c80e938142a5a3adce334f9772bca16ec13b49cb
8128 F20110318_AABRQO woodling_k_Page_113thm.jpg
74723af79ebe2ab479d79ea4c975b17b
5d7488a2128c5c2a04725145d77d1c21dc478336
850836 F20110318_AABSTQ woodling_k_Page_094.jp2
4b222bf40b017a88ba32383f9d31678f
7a40cde63d0bfbff10aa5d30eb0cf5fccb4ce769
952092 F20110318_AABRPZ woodling_k_Page_071.jp2
3fa8c0b258e358c50ff8b439791c191d
876dd5b44dd572da9699aa37c129b8e8001e8a12
96860 F20110318_AABSUF woodling_k_Page_136.jp2
458e3893f98ce9a8b45c2ef9a3773c89
8ab616ca1d6689310bbfeea718b25ef05d7c08c6
94802 F20110318_AABRRD woodling_k_Page_068.jp2
fab6b8eb5e72eb4a4e3f018c8041b2cc
be9c8942f26ddead412c7170d3d816ff282ac94f
2227 F20110318_AABTAA woodling_k_Page_101.txt
3df454d900e0400f6fe682fae34c89f6
d4fb46bbda6bc2e7a17caec7b31b8b6c397debfb
86041 F20110318_AABSUG woodling_k_Page_019.jpg
4972b116f08068fc758872042a464c6c
64b4ade673ad57a0a22f0f209162607fba89d10d
97975 F20110318_AABRRE woodling_k_Page_017.jpg
d399dfbb0a0b17acc43c9e3b8ca91987
d7fb5ab0f10ae3d12752b2561c7c8c77685635e9
34303 F20110318_AABRQP woodling_k_Page_120.pro
e5d3e3407b392ec1c34c5db2cac29f77
e16c778269d5c0b4c13c6c38910287800fec5081
8115 F20110318_AABSTR woodling_k_Page_025thm.jpg
f148935ca0dbe8bf99a1ce3c7ad48484
a9f40ddec702ad74f4a4fcf3c21687ef1093dbc4
2298 F20110318_AABTAB woodling_k_Page_107.txt
d3c92684e4dca0217c16946dc8a9d8e2
f7be54c3fb3a984869cc08f721c9d6d842340c1c
1612 F20110318_AABSUH woodling_k_Page_095.txt
3be8b6ff30eb70c8f5acc5209a3e389f
c9107e3478ffd50036195a2651e8fcaa17a32d3d
29309 F20110318_AABRRF woodling_k_Page_079.pro
e133267a0ca79d8b615c49ad145a3416
9af16e88529d8e3a2d638923e6940e8b939cd2ed
907080 F20110318_AABRQQ woodling_k_Page_100.jp2
0ab2c1614a6cfe31cc5f06c44b8c36af
12438b27b26a8e7d8eb17075a6c0954ee9180304
F20110318_AABSTS woodling_k_Page_125.tif
1c96eaedaac2fc9defc46aa2bcd8a190
01818b459980d026c0292aeb8810d6e2b8cf671c
1609 F20110318_AABTAC woodling_k_Page_116.txt
12a82e04122a12a1bd03bc43e31db2d7
02b69aab944828323833f8f8aa6b2c9b791a2002
23217 F20110318_AABSUI woodling_k_Page_074.QC.jpg
0492e4516f7b03c7221cf0bc7dcd2070
a4fb69a68fd199df365253ead1be6fce2c68cae0
30626 F20110318_AABRRG woodling_k_Page_131.QC.jpg
acad2e5d2af64c850069fa924f03b8bc
7433b4ddd6f7b71457e6a6aa9f4d978ca2bf7f5e
1760 F20110318_AABRQR woodling_k_Page_057.txt
2458407dba70693ffe971f1dc51c4967
669baeb066d2a8d52cfdc81a4e086cd641fd1051
101601 F20110318_AABSTT woodling_k_Page_035.jpg
ee747d4bf7bb4d8031a01094192be166
c19c44efda6079966d984982da77fae5bfac765d
1754 F20110318_AABTAD woodling_k_Page_122.txt
fb77d60e4963badc0276744b041d8dd1
87f6dcfa8fd5d77fca7b43690254877ed2837a75
107796 F20110318_AABSUJ woodling_k_Page_032.jp2
20a714a2aa5b4f9cf615900660024a98
8e9edc79de93960c2a7cb3426c29526c1d7b9360
1578 F20110318_AABRRH woodling_k_Page_052.txt
40f064171ef7a3bdd9361e453a203171
ad23c77b359686682bd6c4908a21413f8f2745e2
F20110318_AABRQS woodling_k_Page_083.tif
df1efdebe8c035d3c3ff3ca2991d9a61
18fa802781c2f7d73427b457f66de23de22cef60
1662 F20110318_AABSTU woodling_k_Page_081.txt
696a0037099c55b425c3852b6f8c30f2
241b2a9c23bed3d4c548482638f9688c1f601e3b
1482 F20110318_AABTAE woodling_k_Page_123.txt
19ef2af4157865b5030c1e89d347fac6
a1459675b50987176bcada433c7fcbf275b05b75
7828 F20110318_AABSUK woodling_k_Page_109thm.jpg
c710b7200cd54e9f2eee7d2cf27e2d89
9305d4f1bbd0530e829e9a79e88e642c3c67b377
1051980 F20110318_AABRRI woodling_k_Page_086.jp2
6fa0c9fe6fbf9f88ddd932d920aff105
a6b6450175a6985e84b995b530516f15441503de
30854 F20110318_AABRQT woodling_k_Page_078.QC.jpg
60909fc80063bf7f5e07ab26a7d973f8
1900eacd8ead554bbdf495e145331b10ac29f735
1665 F20110318_AABSTV woodling_k_Page_100.txt
d4e490816714531ba6be48013e02fb87
43f766292d8c5993d485e5362734b89e3b02b66a
1787 F20110318_AABTAF woodling_k_Page_127.txt
075d754a23001a0714c1328f4e1ec3dd
db0c6ec157b681dfe01f9755b8712d04ca598aa4
76789 F20110318_AABSUL woodling_k_Page_058.jp2
e2504630a026c4413ef3cd6db490c5b0
3f200a1be785bc812789e5a57daa2ab386817455
1961 F20110318_AABSTW woodling_k_Page_085.txt
5c9ca543b8bab8f2544c98ec9662d994
93b8d798371d06179b9516d5886270d1e9f15549
92125 F20110318_AABRRJ woodling_k_Page_091.jpg
29bda54e8e6a6454e24938117e3068d1
ec6a8dd3efa9e8f101fefe4d72f234b6fac73c6f
F20110318_AABRQU woodling_k_Page_096.tif
b58754f8040dec3820fcb9443b1247e7
07c88a32d83bf61216a30d5d1bcf410b13d30103
1885 F20110318_AABTAG woodling_k_Page_131.txt
9249aa5759742c5e48f5083ac3778c6d
5ad65f7f81fed764899a3abab4c7f35ba1b99377
48053 F20110318_AABSVA woodling_k_Page_113.pro
ee1f2ada95fe1ddfaf52d4aa869bb170
f479f7052e419d0b6ed774f0204fa0fe8868276c
29067 F20110318_AABSUM woodling_k_Page_068.QC.jpg
1c1a97629b69ec07d5d80aa5643c4a7d
92cb9a9d013af211cdb025d4b528a4a845a9c2e1
96323 F20110318_AABSTX woodling_k_Page_063.jpg
d4599d10aeb0a37fc45662b061a5206f
b7e5300f1dd30e366adf5313271a10c64a22213d
24647 F20110318_AABRRK woodling_k_Page_094.QC.jpg
b34ea10f8566c698a8f21d9a70c4ced6
8c805f8d317f51a62fe8886235a97b1dd05dad2c
74655 F20110318_AABRQV woodling_k_Page_121.jpg
34a1d287d938d3ab81f9ec08ae7491ed
97f6673c75c26c4ddcee917b67a63b6774c09bd7
2131 F20110318_AABTAH woodling_k_Page_144.txt
75f848335c6302e0623436a4d2187e51
615fed123ce6eff35b61389a16109491630c6fc1
F20110318_AABSVB woodling_k_Page_043.tif
74b4fd28c87242b3600bea695c890b62
734ffc4c53eebdfb295d814115fa33e8c182af51
8546 F20110318_AABSUN woodling_k_Page_086thm.jpg
a61c11779275d46a9fc80d69122f52f5
1e8387e5a3120accacbefc20cb38cc2085a18fa4
85308 F20110318_AABSTY woodling_k_Page_057.jp2
feb01d40ae2bdb464096897ddec0b413
fb768c7df85421a3bedf424fbceb9bf28be41357
73007 F20110318_AABRRL woodling_k_Page_046.jpg
5ccd77dd3f08b98e5201a03a42f89736
fa3d14f95577b5072a38c0979e5cc93d06657a8b
26359 F20110318_AABRQW woodling_k_Page_057.QC.jpg
db79287d85912622c3f5f31539b121d1
1959e069862caf7a451b3df919e1efb930a36829
861 F20110318_AABTAI woodling_k_Page_146.txt
760b237510d15a6b2bf5f9977b3f4305
4ccc1c2bce749738357b1cd453c974bc5b7462e5
31430 F20110318_AABSVC woodling_k_Page_043.QC.jpg
c4a397d071c15d1dbaf8cfcc32e45caf
0ff0bc98e70c36c68195324f8e430b2519f44c71
2052 F20110318_AABSUO woodling_k_Page_087.txt
30815264f57c60dda699d6bed0aaf7f4
66fdcf84ec8d8fc9f028a9328534ceafbd3639ea
F20110318_AABSTZ woodling_k_Page_035.tif
ad273df4cfa68b5dcb7518fa021e3004
30246441011d349db9ef4b3ef3f8f68a35066b07
98480 F20110318_AABRRM woodling_k_Page_141.jpg
2f22fc280b2b80f6d5f565cbe365c6db
3003918bb482e67d78be8452e017efb4fca261a3
F20110318_AABRQX woodling_k_Page_046.tif
c0f5ebeb205c657c31e63ffdaff32d54
4bffb4b3280a5de214d8acf0d02c21cf1c040801
30701 F20110318_AABRSA woodling_k_Page_136.QC.jpg
a5277cdb35550a8a5c3209f734e45073
448c035c9ee2540b8e2f545eb355df08f572217f
9502 F20110318_AABTAJ woodling_k_Page_001.pro
bee3c69cea3a650c262d85308b1dac21
5da28a45567ad8b3158e22359ef11a9abedc364b
842137 F20110318_AABSVD woodling_k_Page_081.jp2
20698db0d3b51d6b8d3739158ef28114
ccb12ade41e53a43ab903d24b3569fc0bc8cb564
F20110318_AABSUP woodling_k_Page_037.tif
b5380ef17355d665484a06194c978033
42ebff1df862197227799e06319b60c6de5e464f
72147 F20110318_AABRRN woodling_k_Page_120.jp2
40892d9357f3d8904467faf2ab3f91dd
eebf6b6c364f641a09d02baa73d1d9db9c19e6a6
31638 F20110318_AABRQY woodling_k_Page_126.QC.jpg
8080c03847a895f18f13a6bdb1b23277
47077979898ad095bfd94e03e55036878b2c67ee
25478 F20110318_AABRSB woodling_k_Page_135.QC.jpg
50025488951e788464d29055046cb5ef
11b33bbc40fbf5c15546357ccd030376c20eac3b
886 F20110318_AABTAK woodling_k_Page_003.pro
80399d98905cc670b1f8a8255782a390
63d15e9b12b5d0675734071f985b8b2175385aac
42920 F20110318_AABSVE woodling_k_Page_117.pro
c40be72ae5ca995b0e55d94850a18f22
85a0acccdb745d5e4e0fb4657774ce615deed581
F20110318_AABSUQ woodling_k_Page_110.tif
d5e99b8b73027357f0ce27af5a681b6c
c8cbe4ba3d8f89d13becbd5969ab057a2888f3f1
1475 F20110318_AABRRO woodling_k_Page_082.txt
ea0570fd059197f2469e9ca50ee87a22
0053dd8889d8102c58fe341a19f483cd98f9c787
28938 F20110318_AABRQZ woodling_k_Page_004.QC.jpg
263daaf58596a09f41117c6e8135fcc8
60ae136924280698ac7cf39aa035fedd648a8a9a
1038249 F20110318_AABRSC woodling_k_Page_078.jp2
3c2587d8c9d6e21bacf6045c08931769
2ca7b1a3a1b86d30bb6405df05ab740067bbf869
42134 F20110318_AABTAL woodling_k_Page_004.pro
73d90baf85b8749eb40c5059568f32a8
43468edf3beab8702151e06e699148e980d9a4a3
45724 F20110318_AABSVF woodling_k_Page_109.pro
d3e2ee21a64b1a7ba83e54ee577c01e0
0a446b78dba3d7c0e5e874dac599223c16b31eac
50332 F20110318_AABSUR woodling_k_Page_072.pro
273a0b336bd062c2af9105386d1f0e71
189bb63b7b7b6d5efc32e3c3b6f45da7c655e613
8383 F20110318_AABRRP woodling_k_Page_020thm.jpg
b20cc95e03613f52fad0bc0634302da1
89cf5617f3044afb0c00fc400cd76be4159f67bd
F20110318_AABRSD woodling_k_Page_116.tif
5997c5dec590bb93bd3d91af268d05a5
c478c75383920426f378b90318d648ebed0c6c70
45416 F20110318_AABTBA woodling_k_Page_128.pro
0af95402a422471536f8c5d63f1128e5
3dfd32a81f2a404ce8f8f451d9c462df951346d8
50811 F20110318_AABTAM woodling_k_Page_028.pro
2fc639bf4d22e3aa5ed0cb74e45ea225
94dd068e422b7fcf9e8dd56395d6144458ab4d2f
F20110318_AABSVG woodling_k_Page_057.tif
35182f356d5b8e2dc3b002e45bc77c4f
f4d60066e024b46ca4d76ad12a09c778f5531b70
F20110318_AABRSE woodling_k_Page_002.tif
224ec63b5fc17bc7e639c573b11a8e69
8eb80bf460adac1670619fffb1b5eec3d680054e
47629 F20110318_AABTBB woodling_k_Page_141.pro
dcebb1dcd02929b360b24970d60e3163
cd69410db475570e509f588baa8f40e64cda7797
50468 F20110318_AABTAN woodling_k_Page_031.pro
bb9ebeb959ea9ec55971a225d251063a
3587bcba78d3829b70d271a60c1f818f692c6558
1761 F20110318_AABSVH woodling_k_Page_055.txt
5941111aa302a862601e2e084373ae8f
a2d17d02d9a84026d75440c9bb6e03ba5df6e26a
1878 F20110318_AABSUS woodling_k_Page_063.txt
2a9cb569ae7c93c0260b603496451863
9c8f59ff4655ff2b618a3670cea556de8fe1ba43
26036 F20110318_AABRRQ woodling_k_Page_122.QC.jpg
dd8cf347febc426366c6ce235dee89b7
56bc01aeedf58d6fa6db1609731465467870fda7
35856 F20110318_AABRSF woodling_k_Page_051.pro
73577eb67d19a6170a3e51ce59cf8c8a
13a7c9ebdec1bec791b01d0524eac57e87a5d519
107098 F20110318_AABTBC woodling_k_Page_024.jpg
9e87681e6aa92ed51221e6673defde7f
6ed080bfeeb2eb3ef3fcfb964e33fd45b45d3132
51196 F20110318_AABTAO woodling_k_Page_036.pro
33101770dd7da2bd456fe8cdfb1a2da0
b798e9cdd951a6c3466ff580d3bccfddc8b9d64d
488 F20110318_AABSVI woodling_k_Page_008.txt
1c350e3197c0b105ea678b53367b3a74
692824e85d941cac0acd5e76059e614bc1f85e7a
24529 F20110318_AABSUT woodling_k_Page_077.QC.jpg
39bf980a4da85b27b1b05a1201ccf794
c49c6f577d58286c6cab1496e78e9fc4890d68bc
98675 F20110318_AABRRR woodling_k_Page_128.jpg
49e2f0e2d65f6e818dff0119d56b5a2e
af12b94b476fb3fb4db5165d69c0983e40def4ec
7917 F20110318_AABRSG woodling_k_Page_073thm.jpg
80afb719aa90939489d8d4f1173f3580
3c017867f770d9b65b2894f0cc533022a89e3e71
99337 F20110318_AABTBD woodling_k_Page_026.jpg
19ff7c38d2f62ef7eb0c2ec96e1a675a
3075c2bce2db049ad32737b32ff33ac4bcc51c10
40400 F20110318_AABTAP woodling_k_Page_038.pro
34dc4965b5cc135c0897c501e51e6eec
27e47f7b341956b89378e2dbd36b4fe3f01326fc
F20110318_AABSVJ woodling_k_Page_075.tif
376ec9aead3692cdfee282e1ae2f939f
a63baa0f2c1d30515b8673ede53c9f864b677cc1
32898 F20110318_AABSUU woodling_k_Page_054.QC.jpg
60e1792ab8e2610dc1c114e6a67fd17e
81076d25616d6fc255f4625fbc927fbc83f36204
6302 F20110318_AABRRS woodling_k_Page_135thm.jpg
1261cdab906f5a399da8ab1fabd1731e
6a1b8749f7863572e46b02c883faec009337b063
93441 F20110318_AABRSH woodling_k_Page_125.jpg
527304fb2095e962f7b08ab4cf488a48
c0cd473de94d097dbc43d79c7a3834871994b0b2
101345 F20110318_AABTBE woodling_k_Page_039.jpg
0f8bdbc60da4b712250688bffe895156
c9f8291f53ede2500e45c2fdcdb65adf451f3368
51552 F20110318_AABTAQ woodling_k_Page_062.pro
d9d9ef05a6e222513a29bbc7cd3e59e5
bd6d63b2870960fe5af3c62f7427ce9ed7794e94
74485 F20110318_AABSVK woodling_k_Page_123.jp2
ffaf3d2ae0ba1833c2fd43b65e38a397
db8d99214059dc70e652da32fbe233342745fa75
86580 F20110318_AABSUV woodling_k_Page_042.jpg
a6359765a6a29e651877a6f6fd87b274
906f139eb8da7c94093b0a20034ccfb36c7f75f5
30733 F20110318_AABRRT woodling_k_Page_138.QC.jpg
94314499b63f8e51b30e4279ee66a8df
1835e43a00d934bac1d6c36038722f84ec88a1e7
101442 F20110318_AABRSI woodling_k_Page_054.jpg
a3cc89db524e1b313f84054e8020e1b6
304425034f2806acc3c3ba550a91837ce327f6cf
69764 F20110318_AABTBF woodling_k_Page_051.jpg
5b543eb6b98f775c9ceb14336c63cf4a
e308bc919479984b5159c5d5689a2b696f71fc67
49560 F20110318_AABTAR woodling_k_Page_066.pro
7ef65cca0e06a7d68a707c6310b28202
c0d28d8ab6894b87be23e6e94e8e24ec5e539dcf
1817 F20110318_AABSVL woodling_k_Page_139.txt
82c5407b0ee40c8ffa46d4804769154d
1abcc100bcc0450a2d88f7ea9970d242ada57ce2
8402 F20110318_AABSUW woodling_k_Page_134thm.jpg
0581cad6e35207c5393a1f9c2bb7fcda
678d3a27a7184cacbe6ef1a31fc4e2bcf7d53a74
126968 F20110318_AABRRU woodling_k_Page_011.jpg
9d619bf4ffb92b2a843e3f05ce17f1d3
385572323294ba456010419992645e7755bf5c90
53388 F20110318_AABRSJ woodling_k_Page_044.pro
82e77fc7ef319968fee5f406d87dd23c
0b970fd482ff9b7a07947a00555f560a92b1ddf1
83555 F20110318_AABTBG woodling_k_Page_053.jpg
eb759b34d101f5ccf1d034626eebf3b4
b99a3fc2e484d53895adffb8a55e51bf5c2c9b01
42495 F20110318_AABTAS woodling_k_Page_068.pro
1720834b9a2637d5a80cc0c64fe80b85
3b3717008febce3d978c716ee33deb4377e0e3f0
8370 F20110318_AABSWA woodling_k_Page_126thm.jpg
95525e69d9a253f44367fbe3e4d62ea7
b8ae0c66ae862480c0121bcf7b1eb926fbfad1b6
2246 F20110318_AABSVM woodling_k_Page_086.txt
2f981c9a238dc753929bc4cf1630c74c
2bcf8ffaa3b91cc7fa1df89a1853cda93c79fe31
8164 F20110318_AABSUX woodling_k_Page_115thm.jpg
98cb0bd31dc69c72818393f10ccf3e6c
af5a7e03637b50f7b2473849341a2667148bb10d
33629 F20110318_AABRRV woodling_k_Page_107.QC.jpg
9b5172b1d434b7381cc20d91774e33f7
d0b51ffb2febba8e11873306c7498b5cae9ec700
F20110318_AABRSK woodling_k_Page_022.tif
0ec2dad74b41e86337b78ef042c461c4
515f711c6abb8d58c9e587e3cbb4bab506e75aae
89379 F20110318_AABTBH woodling_k_Page_059.jpg
2c5d17c68612769890b79279e16c11b8
da0962c2bd911cda0328ca1e1e7f9edbc5582a78
49108 F20110318_AABTAT woodling_k_Page_078.pro
685c9f576dc424440010c5fb6ff81409
4268ec365bd07b6ac1df0bf288b6c5c30fa4804a
49726 F20110318_AABSWB woodling_k_Page_085.pro
4b8cd157914c96b091102bab64afc8cf
04997e2946c55e85f8bc61717fa1fadab5a61fff
2237 F20110318_AABSVN woodling_k_Page_054.txt
59df307aa95dc4cb6efeaa2b639c07f4
13f0cbb75c2437063a7068865a97f6906c54ff57
8465 F20110318_AABSUY woodling_k_Page_035thm.jpg
d11bbc03177711228d864879e0456955
b8054ccf6cdd049dc6a76e32acf369bbbfb0c348
100061 F20110318_AABRRW woodling_k_Page_060.jpg
742e3150cf468d5de5f39d1e721453d5
84ed982abd58a760b14dac42c318cd71457cea19
100779 F20110318_AABRSL woodling_k_Page_063.jp2
a5215824611e3ebf5087410647b6079b
15572509c02cfb39298b5d282c42aa3b21493955
104629 F20110318_AABTBI woodling_k_Page_062.jpg
e30fbf1bdf7c98a6234fb82fa08dde29
eef160193b0d9e50e14950891e1bfff1c56e66c5
34132 F20110318_AABTAU woodling_k_Page_080.pro
244db0bb1171e185148995ec49c416ac
8fed63d60acdad6b7ed2a9e1f8563514c993fbe0
31858 F20110318_AABSWC woodling_k_Page_061.pro
418e9fb3556a667b10e9dbbee18c67a1
8abc04cb4636e62060d21a7589a8ba0c70370dc1
F20110318_AABSVO woodling_k_Page_018.tif
1e3a5c852fb456e40093422e31a42d9d
f03fa8ad1d4302614bb935e1c7f4a17cdb34d40c
21252 F20110318_AABSUZ woodling_k_Page_130.QC.jpg
132c292bc913ce922bb029e0df3aa2f3
9904f3367965c1ebb888f50271060dbab71bbe0e
81117 F20110318_AABRRX woodling_k_Page_129.jp2
d913469fb285b32a6b9af2a53c4d3c2b
ace50d928882df6310c739badf7a7cce7e377ad4
96846 F20110318_AABRTA woodling_k_Page_114.jp2
cd221f3abacf6fc85a3c716b8eac56e0
00fe52a16384bc0128ff8cb2998c3f07b0c5b247
108568 F20110318_AABRSM woodling_k_Page_021.jp2
db118920af434c53bece768f52f580fd
52095bf8a4fe7a2b6ebbf294151ae757a60875fa
60934 F20110318_AABTBJ woodling_k_Page_067.jpg
b74d6f35680a2022ce561aa8986dacb6
0e99f14a31ef5a30370f4dfde78b6ef8d400d60a
53638 F20110318_AABTAV woodling_k_Page_086.pro
61bd8c1338cc2fb355b650e3e4fcf314
c871c16369e589d6eeea67e22d5911bf3511e2fb
886851 F20110318_AABSWD woodling_k_Page_111.jp2
d17a83a80099cbfa1c1947c889feb0dc
c3ce36fe2a2b03d6308ec1ec5ede546b713178c4
106382 F20110318_AABSVP woodling_k_Page_007.pro
0441cc8263dfedaeeac2d7eff60ab8b4
229607a719aeec599f3e360d13cbfbc6abab99a6
108360 F20110318_AABRRY woodling_k_Page_088.jpg
69a6de0a07086f8e36f87b06b3d23335
a79c587192d20254ce8974f17d5e31867aae4f3c
29281 F20110318_AABRTB woodling_k_Page_116.QC.jpg
e59b2ea5643c15a6dce2c2bb6dcc7db5
51476c424839d178f809d94f5ff82a92a2f718b4
105126 F20110318_AABRSN woodling_k_Page_060.jp2
8e12d1a2ee43a62948f57ed5bfc197ba
0894dc2f8fb7a9f2d22cbdee19af98a3ac0d5a0d
87601 F20110318_AABTBK woodling_k_Page_070.jpg
4daf34c2e6bfcf9dc14495f1c778b86c
79e8cc8e097d9c9e46d9490ba4412aa087f60e11
42969 F20110318_AABTAW woodling_k_Page_090.pro
fcce74ec4cb6c79edc35266e75214edf
0ce0ed5ce63cdba75f67cc6e582b5ccc1bad1d19
107706 F20110318_AABSWE woodling_k_Page_118.jp2
876305e0e91e481ee5ff37b6bce848b5
0f7e3cbb1891b94c18e0b4e2636efa19f85c2965
8435 F20110318_AABSVQ woodling_k_Page_140thm.jpg
cc821ccf1d58427560bc0c8d9b56369b
642e25b7e8e37961690704d64c445467b71611bb
2080 F20110318_AABRRZ woodling_k_Page_142.txt
2bcab4cd60c71ae346471750ef06ec66
4c3651497f82779a8faa10adf0d4b65b39ba2e80
6616 F20110318_AABRTC woodling_k_Page_093thm.jpg
b4155cd594a9d785678bfaebe8fe87ea
caae9bb8dd470ad589236c79e84fe59a00064f1f
F20110318_AABRSO woodling_k_Page_105.tif
31fcebbb22cb9e67758a76e0d9efe60e
e83c0e77bec68632ccb9cbab7441301b0de75b5c
88983 F20110318_AABTBL woodling_k_Page_071.jpg
2ee8616bfd938b45c0dc233bd712dee8
56f9c66f0de0e3efb63235908a13a741aa8573ad
43328 F20110318_AABTAX woodling_k_Page_091.pro
3f1b4ecb29cb27c135555532dfd073ab
3f57f45103fac1472e1a685511530ef58728028f
F20110318_AABSWF woodling_k_Page_127.tif
a1f3c914971fcad0c9079208219450ee
04ad2614721ee69a3ff6dc0c9a745cb34374ef44
7881 F20110318_AABSVR woodling_k_Page_103thm.jpg
7a95415cf0a225f61aaec082592960ce
7901fa7d02b51cf7669334fae4b584c066594f71
956799 F20110318_AABRTD woodling_k_Page_103.jp2
4391f978fe12d8e8c999a1af7b2f1282
25e073e04a4ad3c6f8f0e1e6f8ee1c80802f9ed9
F20110318_AABRSP woodling_k_Page_081.tif
acd191b6f7bae548d71b40a2abb9e13f
c973e72a1eb92224ef61f5096b583671842ed797
93720 F20110318_AABTCA woodling_k_Page_084.jp2
c5aa2fae07a70052749fe0b393282b9c
25ea4d6da05d6c66083a3fdd0e6162f002279308
108143 F20110318_AABTBM woodling_k_Page_087.jpg
64c2c577c38b7beb80fb73e5bc870383
adb366aa476916dfeeb59d7a0800baebabb12b6d
47835 F20110318_AABTAY woodling_k_Page_119.pro
ed9fd932acce021173827fd0a6875daa
b853a339e672daa41b19b2748db503df0562b7f1
4011 F20110318_AABSWG woodling_k_Page_146thm.jpg
71a357e078301fc555ff145ccab7d1aa
5bbeecdd463684b31c5948379cfa061a053fca24
1632 F20110318_AABSVS woodling_k_Page_092.txt
4836f489f86e803e2d39ffc5779f5c85
cde48a72cbaf4f5fca52303d1d659aa5acdfd5d2
F20110318_AABRTE woodling_k_Page_094.txt
c583e73cc7790dc5621375645d14c846
b4d993419006d59db9efb0c2fa689cdda5bada68
27210 F20110318_AABRSQ woodling_k_Page_071.QC.jpg
8fd9cf715a76ed0eb4f07a3319a571f9
8e0ec05deced79f33871e1da1628d06bd87e0190
109661 F20110318_AABTCB woodling_k_Page_085.jp2
c59d331ac0f6ee5617584d0bdceae5bf
d97e183e784f5f47559369e234e4458975e723f6
94463 F20110318_AABTBN woodling_k_Page_109.jpg
c5e430988a07be076cfb0e5a7d61e29d
5f3d546c37f2d08f47c9013175126378496ef464
37522 F20110318_AABTAZ woodling_k_Page_121.pro
9e2b06c6a268bf5d4e584c5c93f0e377
425cdb0dfb66ddef79401de186fa43bfe69b4fa1
99593 F20110318_AABSWH woodling_k_Page_018.jpg
242cf38bfc2e003773a086a6cc5c7f33
c2cc3f385339a7c74bbc1b8d5d8222de1d3eb8f8
29821 F20110318_AABRTF woodling_k_Page_010.QC.jpg
5ae9eda8d64d3c8381d78c8f0c4c778c
7c9cf5feb9a8028f6a5b48ac9164675479921966
973217 F20110318_AABTCC woodling_k_Page_105.jp2
96c337005c85418563375084a0b10083
0d0ba6d8ba688b56dd679657176ad48db9b384e6
97834 F20110318_AABTBO woodling_k_Page_126.jpg
0134b85b1dd5b569e67fe4d9331099f7
00a483f1064c7e51b20fe8eeb3e7b5129924e479
837302 F20110318_AABSWI woodling_k_Page_122.jp2
f4c7c0963784d79ac5ca9c440ee84022
c472f65fda2c4509f4f28b8556b9c49378152fa7
F20110318_AABSVT woodling_k_Page_068.tif
b3b2211c16c128cea9900f716ff47fb1
4c63487a793e99c95a1be17161cb5b6fd7bbe64c
648091 F20110318_AABRTG woodling_k_Page_067.jp2
1f33c11065116fb941bb8681c3bcb7a5
b998dfab732bac2b3a6aa37de98bf56241b18ca3
1783 F20110318_AABRSR woodling_k_Page_138.txt
bc949f8cea796ee0e8a38f33f5bdd1c3
1ebf9e8d66033291c996b032c7210cd641c0aa7c
999916 F20110318_AABTCD woodling_k_Page_109.jp2
45641098c5437b29970b775cd2430690
9c36c4d409bddeece022c0fce848856f4bf221ca
65862 F20110318_AABTBP woodling_k_Page_130.jpg
4ad0251e8a1bfabe61588cf39d124975
89c2df2008cb829a490a7c3a5f488eca51a6331a
8316 F20110318_AABSWJ woodling_k_Page_032thm.jpg
b12e09388ffe5181f2c8232f3f9d2e4f
75fc5703aaa67fec4a3c25c83cae2b77436eff35
F20110318_AABSVU woodling_k_Page_089.tif
2b3f4ed27a6bc0fef621a32d24e103ab
9f124c42083db8fcca5fffa2a66bf38187fa0769
F20110318_AABRTH woodling_k_Page_045.tif
131312e26cb6477197dade3bb74ceabd
54c53c562602fbd5a8eaf120aba32cb5b06353b8
1051985 F20110318_AABRSS woodling_k_Page_066.jp2
806407b5917dc6fa64b78df10c7d746f
a79c8da04e2a5d4f2f7cd8e580bca06b8476855b
968399 F20110318_AABTCE woodling_k_Page_112.jp2
2d44b32a34c8d756505bf593d1e20003
29d0e972800b0486492aa94100f9e840d3b75ef9
102525 F20110318_AABTBQ woodling_k_Page_132.jpg
a05efc63c9df265413cb7e22b0b0ceae
3e6a75f1a0a7ee76121ccd22b385f24eae670c98
12585 F20110318_AABSWK woodling_k_Page_005.jp2
c65a26258deaadf5b6f72e4c8b2ce668
8a07548c2a27ffee346a40ee22a98b3e38ab4189
2118 F20110318_AABSVV woodling_k_Page_143.txt
3cb38c2ee62b20b78b6cdf6efd00d8ee
082e334cbfa9eb961d426523991dc3cd62926bc4
2139 F20110318_AABRTI woodling_k_Page_109.txt
ff341a9efc573c2178790877a8eecfe7
7b4a0e9d49c0a804eaee2d7931f599ee4e84b718
27425 F20110318_AABRST woodling_k_Page_100.QC.jpg
c098c7dc4fa97d6b8f1d2c2e7cd8d323
b7a0203e1f0288545cfc0fbf681ce45529a0eb67
1030251 F20110318_AABTCF woodling_k_Page_126.jp2
5ec45fca8ae23f8b2aa4d28108ec02de
00854ec855eb20d99ac9fc2fa99002200cfdbfbe
1051973 F20110318_AABTBR woodling_k_Page_012.jp2
23a635f296b5ba267c0502cce6d4c718
e6c1f3fae6038dde33828f412f7dd0696994c8cf
42995 F20110318_AABSWL woodling_k_Page_033.pro
57d4ed123faf813679fb3dd9a00b59ad
b8b839c066a40d21922c49deac8ddf92ec3868aa
33570 F20110318_AABSVW woodling_k_Page_037.QC.jpg
c8f05328f3632d1a14692f8dbd51df58
e590dca54f0b48e7fd78907a4158fb8cf1cae5af
101819 F20110318_AABRTJ woodling_k_Page_069.jpg
9f8a963915af820e4c31312845f1666c
d37868c46ad3feee10d55532acf047db0ec289f0
70334 F20110318_AABRSU woodling_k_Page_049.jpg
00d4298e0eac25a73e1b76a418adff85
c16082adb4c30638b1b44212f678dd2eb67df750
99125 F20110318_AABTCG woodling_k_Page_138.jp2
24b16b2dd74eba8605c1212f2baa5860
0bc7adba3afea13597d13ace3ed947e494e6d045
105646 F20110318_AABTBS woodling_k_Page_017.jp2
df8546fafc6cb17c9d90ef112020ce81
8f65457178aab292f27ae347f39cedc6841af0e4
86414 F20110318_AABSXA woodling_k_Page_014.jpg
bf4153a089e41fedf702ce558f16fb50
cb035add952dedb4c1ce5db00ab80bb5a61e5476
30696 F20110318_AABSWM woodling_k_Page_130.pro
01ad3dc4852cd51781d215cc39f016f2
bcd0f212ed065cc762d7a110af876aea014826f3
103470 F20110318_AABSVX woodling_k_Page_036.jpg
e7b020e54b2ca8c5d7bd194cebad9011
4c9cf1c8d84005017d3dd6e45e798d267650594f
F20110318_AABRTK woodling_k_Page_134.tif
e488a45f36ea61a5fbff56d00c9728e8
5edcbec8e16c9fa2b17b957d4b2bb494011bfcc7
515 F20110318_AABRSV woodling_k_Page_001.txt
bb992e10646e04b63e850060fb87793d
4f9509e8e3d548992d82282ad2141b78bf5f9645
4860 F20110318_AABTCH woodling_k_Page_067thm.jpg
39e275e350cab838295ac68bdf9e154f
0f0f9b43df0983c0ca5967a3ed9848880e9a7a2c
113704 F20110318_AABTBT woodling_k_Page_024.jp2
dacf6adb48322c85ba39764f6e03ab79
e81777f55d0d1e7d6766710dfaa01df8d6a2bcdc
32515 F20110318_AABSXB woodling_k_Page_060.QC.jpg
12b32efbec6edcf0fdc17bf4072cd755
26a3ee05368d5f779e059f26a6973d22ed055bdc
1999 F20110318_AABSWN woodling_k_Page_090.txt
78bf9d6dc39d3dff5ffdc9dad0787e3d
6b440e21da9eb5fbc73a46147b0faff253830652
31476 F20110318_AABSVY woodling_k_Page_128.QC.jpg
bcaaa95b31b7d25849b930b61e230c98
f875ebf27e1888fa03c51a8591503d65d7739b98
7997 F20110318_AABRTL woodling_k_Page_139thm.jpg
0e6d794e9360a1199414788dbc2949fd
b0e67272f482be2cd30cadb614004b177f024ad7
49184 F20110318_AABRSW woodling_k_Page_118.pro
bf9afb549495e021eda09a89ba86e3e0
052fb19f877f01e8c86686f3026f39276cd67e04
24413 F20110318_AABTCI woodling_k_Page_129.QC.jpg
52c2a77978f632914c2f31276db56f6c
91a8b9419bee6c5bbbb1a3d15b2df5842e05b2d4
106204 F20110318_AABTBU woodling_k_Page_029.jp2
ceee9108465612c0a87ce6976419cd4b
b362f02fd74e6b7538dc2db3b125e338eda62977
5908 F20110318_AABSXC woodling_k_Page_015thm.jpg
8a868ddbd8efd2de5205538c38a543cb
3d7b45595ab5ce76fa72ad560baacbef7057c995
F20110318_AABSWO woodling_k_Page_133.tif
935c9751951be7a85d8e961aea969cc4
f0f6d977aee7c5661f0587aabf1bea7a8c2c97b7
30273 F20110318_AABSVZ woodling_k_Page_103.QC.jpg
7866cd17df24b2a43bacb2ad4346d800
cbc5626a324a19d4971cdaf52d55a8436fc88b2e
8059 F20110318_AABRUA woodling_k_Page_030thm.jpg
a836cd954b8eaf2380ef5e269a8ecaea
641a4f8c799c03247d53d1bdbfb19f688fbf21a6
69309 F20110318_AABRTM woodling_k_Page_034.jpg
f43a079623a346e31d7c957679b25a08
93b8af4f7d698b54538cd308681b4949ba223102
F20110318_AABRSX woodling_k_Page_093.tif
ab655b0ac79ca78d52e3b9466cea4563
c1d2298ff30427da46c1d573ddb742852ca6a7cc
7252 F20110318_AABTCJ woodling_k_Page_098thm.jpg
c5ba4e20df010d2c37730a4b431286e5
2b1c2b34e6b9c949042e7f1ef782b2ada2f85df6
995855 F20110318_AABTBV woodling_k_Page_033.jp2
a723ac1a53d2a575641ad251663e4f7e
f3b663dad52ad66f2d44e94991c9f133a1ea7074
32959 F20110318_AABSXD woodling_k_Page_029.QC.jpg
257a48e697b167e1e638e43c5ad98bf9
b31161bad4e1618c6d3d73442702ada6813249f2
F20110318_AABSWP woodling_k_Page_065.tif
ebef464274810fcf134ee596960ea325
7dfc287eef9287e0b7c3ff0a834b7b400f9efe93
40103 F20110318_AABRUB woodling_k_Page_125.pro
f3e47f896790e01840d47fb5507c960f
34f0983eaf481a38255be22d096996dad597de96
F20110318_AABRTN woodling_k_Page_059.tif
764cf50e70e323adc8718352c4235b3f
c7ede98c2111a52eb4049cf1e8fa8e977f914bd7
96508 F20110318_AABRSY woodling_k_Page_059.jp2
e7fac6db05160ec6ed592ffdd5ec463b
b59f7b2d0c8c2b3d8591dfac006f77264219763f
7754 F20110318_AABTCK woodling_k_Page_063thm.jpg
bd609551e92b7ec88c403e65b013e6bc
6ca0a0905b683a17997615bdce6533bd44113751
72982 F20110318_AABTBW woodling_k_Page_034.jp2
7b10fad4fa549e3b119b917c709ff1b1
32a59774109c78c6e1fa82385053aee4cfbfdb26
76148 F20110318_AABSXE woodling_k_Page_015.jp2
4527f8b25b466d29ce2fb7cd3cfa598c
789c1887a26497088fe3c87846b7ca51f8de99c7
31135 F20110318_AABSWQ woodling_k_Page_139.QC.jpg
d90b44950a7763617d2f8b9e22c3e1a8
955f2e3f95ba866553d0f7c82569c45ff467d4f4
89921 F20110318_AABRUC woodling_k_Page_033.jpg
19de317e56ae7c08b5e9c70b8f159969
b487ba0d70f380b3541a3bc164dfeb5bba51fa04
26590 F20110318_AABRTO woodling_k_Page_096.QC.jpg
982e4dcad4be03cfbaeae303b4f315a3
0819ae0c4aa7e6f91a621d51b35e1fa609ee2759
109434 F20110318_AABRSZ woodling_k_Page_144.jpg
baa3f80df0bda5953abadd46315dd55a
63435ab1532ade049c57a49ce4b993288fced255
237503 F20110318_AABTCL UFE0012801_00001.xml FULL
66c29fa2b8e391fd39d1358d501e65fb
d365d0033219bc02624c48030fbe4032f1b4bc92
108849 F20110318_AABTBX woodling_k_Page_035.jp2
ae4a85075240650f51cda05cd43ff964
b0a597ea0cbeb2099832cde556535e35e1daf048
102098 F20110318_AABSXF woodling_k_Page_140.jp2
301ad175cadb2b456de267df8d193613
9719c41a93b9babbc6836d31611566e16e4ec26b
49650 F20110318_AABSWR woodling_k_Page_115.pro
a2156d582d2268613d6c1ba69658875c
eceb4e912e586fc7505cc413f379e048dbee17a1
74584 F20110318_AABRUD woodling_k_Page_049.jp2
399c5d793c1c157eae2eaa6444efa325
4de5291b97365780ce4bac252bba6d6ccb123d81
77982 F20110318_AABRTP woodling_k_Page_092.jp2
3d8918cf58ab30a6d42f08bbeae91360
4800e3dd736988c1474d4cd05450a66b2b94cab9
8815 F20110318_AABTDA woodling_k_Page_142thm.jpg
eb36fcb033d4fa694d8c1240a2db3e9b
2ffad2374e68e4ab9bfbd2a0ba0600789eda3b91
5156 F20110318_AABTCM woodling_k_Page_008.QC.jpg
c744326d179086948d71a005195e3cb7
351ea3aa59284aa0dd43ba2aff31bd8b337ea20d
72643 F20110318_AABTBY woodling_k_Page_051.jp2
b6d219c4bb0642c9eb3251ddf4f68f0e
0e111d824d693e24d22e7722b4308521d0945fc5
27650 F20110318_AABSXG woodling_k_Page_022.QC.jpg
5a6286662f5f509de48d6470a0573f77
e79d0baef40f4caad8e81ca660b4de2a31b685b3
1674 F20110318_AABSWS woodling_k_Page_074.txt
969c21a9cca84c3e4a2ae2ef994af9be
b7f215feb3425f1db5c9af36ab14dc4cd6dbdddc
34535 F20110318_AABRUE woodling_k_Page_023.QC.jpg
4177e4def88ed85058ca4a0fcfe2549e
9eb1fc8b0c39573e09c671af239e41e67d6f51df
79353 F20110318_AABRTQ woodling_k_Page_077.jp2
3afa3e67d117ae93244a61a0eeff08c9
3a7fad30d3403f65bfbca5e1de2b98026c356374
8779 F20110318_AABTDB woodling_k_Page_143thm.jpg
20100c5c3ac85f93ff489bd96e533c8f
6645cfc79db5254795d87f570728804930059f1f
31665 F20110318_AABTCN woodling_k_Page_026.QC.jpg
d0ae9b25774d6c5904a72fe7126784ec
406881beb502fd1998e9fdd4fc89127d244aa14c
1014531 F20110318_AABTBZ woodling_k_Page_055.jp2
9872dc1b20bed5098ffc63f0991ee48d
c8dc2eb07d9d019ec43d7f0339cc9750edfb1fc8
34903 F20110318_AABSXH woodling_k_Page_092.pro
861bee215d027d8f138c978cf1a1c177
cf4b8b0838f9e5335b1e77bffc8b5879b1a77170
172 F20110318_AABSWT woodling_k_Page_005.txt
07f8138a8578c690e43856c33185a5fa
2c4ff5d406e2ee9e53dd8b819483220cd0dfdbb7
F20110318_AABRUF woodling_k_Page_021.tif
50a283831ac2f06171b50efee9d9499c
d1aaa10285358fdd9ad545e7446f4b393fd4f82b
8235 F20110318_AABRTR woodling_k_Page_039thm.jpg
86f0992f39b4277b9ecfdcad87097f55
27c56475caa26c1d8dd64c33b75f27448c2ebc50
32214 F20110318_AABTCO woodling_k_Page_030.QC.jpg
6682832c4ba502ea885e4dd042a81f1c
886c983cd728113a41b0e9f83e48d281dcd7d51c
24270 F20110318_AABSXI woodling_k_Page_041.QC.jpg
723ee777b5e7cd285c1edb54eaae35b9
a14b8b24453404bd70c75a16290dd20c6c130671
F20110318_AABRUG woodling_k_Page_102.tif
2562894df47cb2b0e1030d977655a9b2
fa2729bc04247cdc22933365dc50ad3a9c292df6
37090 F20110318_AABSAA woodling_k_Page_081.pro
0b4f94808f6665ae3076a758bf7b11eb
d3851267e91599171666cd52e39681df075568e0
25632 F20110318_AABTCP woodling_k_Page_038.QC.jpg
3eb4923ef07bbe7e29b3e6a4bed76e4a
ab982cfa33bc3674cc61ac2dc971310b38d34530
1051960 F20110318_AABSXJ woodling_k_Page_045.jp2
ea61b74a7cd3cd7d157aa3625a641407
b1b1d8272d3919c1703c2eea8717baf26675ba9a
23422 F20110318_AABSWU woodling_k_Page_034.QC.jpg
b68b72010a2d69bbeec524018400e973
aff6f378ff80b6a083e2229b9655c78d0a15c972
32418 F20110318_AABRUH woodling_k_Page_141.QC.jpg
3ced6938adca21f6e3fd7402c02be615
9841ef18b12a874d0659dcce6fed4d1c474ad6d7
F20110318_AABRTS woodling_k_Page_003.tif
5af91efbabf9f707ef70290a6691a980
e00e67c00ca533a0d28740dd380d60438a3426a0
34133 F20110318_AABSAB woodling_k_Page_096.pro
06089536335c33cf9c2a439609a9662e
95dacda191d841485afa300874575965290d5434
24987 F20110318_AABTCQ woodling_k_Page_046.QC.jpg
da575609a719681fb77b41ef48414943
855ee0a705015e105910a87f50357b39d246b9f8
36604 F20110318_AABSXK woodling_k_Page_129.pro
816d963d9b38c71927165beea02babe9
1592a63fffe10be64e0776d2edef1ec36dbb0596
2159 F20110318_AABSWV woodling_k_Page_066.txt
ed7dc1334b55a12ff2a8d1e5ec0d2a6f
33626f62be8185b313867ebd99164c74680eefbf
119 F20110318_AABRUI woodling_k_Page_002.txt
3fac8553d71c109c14c9de64152af1ea
20fe899277a61c126d954c2d4317004df9629cf8
F20110318_AABRTT woodling_k_Page_015.tif
1a61a926010ff6b344d8e9e838f16c81
cd0e2909bd3711943f2e4e538cda4854a3fe6fe1
F20110318_AABSAC woodling_k_Page_145.txt
bf63b5d3866a86c04d72d5dec9db71b2
140988894a4bdf503d888b9a14db9406c239d7f0
33001 F20110318_AABTCR woodling_k_Page_069.QC.jpg
172e68f65bff44fe844afd66335601b2
a75b38b4df9ec6aa1908ef1dc4d76595041622f2
94926 F20110318_AABSXL woodling_k_Page_124.jpg
b414084475ea41bf8f985f2eb7f41fbb
55e8e3ee75a32eaedcaddfceeee642ba82c88a1d
1807 F20110318_AABSWW woodling_k_Page_084.txt
3be15067780d5ea0a3108082a1fb2dd7
05369bab44b7caf5a06317da53821d168cae2a42
28814 F20110318_AABRUJ woodling_k_Page_117.QC.jpg
aaef54e949702b2a6191035e9e7ff41f
dc789f39b3a3e36024785d215c0462341e41be2c
49101 F20110318_AABRTU woodling_k_Page_106.pro
074fbf4344c71af40115b0a572954c20
58f39462ca87363f7db29ff08d7b6b086cd54868
50560 F20110318_AABSAD woodling_k_Page_035.pro
5af1f9e7621a214e3b36e09636124b1d
f789144dc9aeed29f01b7ca9d66ee7adfcbd6e6f
28604 F20110318_AABTCS woodling_k_Page_084.QC.jpg
820309c426ff92c9798e1b57dea2e67f
9a90ea37a9d728e5cab4303ee1eaf4409951a142
6741 F20110318_AABSYA woodling_k_Page_121thm.jpg
8f912b05f3c8f7b0def71d061e7dbd86
7201a2dd7946ae74aa4604b39d904ac22fb036e1
86795 F20110318_AABSXM woodling_k_Page_100.jpg
5e6c0eb88852dab93b2d9f390bf41bee
8c9a68e28205ab9417a0bdd0cfdf37ce5b85c065
2066 F20110318_AABSWX woodling_k_Page_024.txt
2ff37ddcddebaa2d0122b99a0499ffe5
4d34a546fe2a601fd0908e69d351a3feaf1c4516
8593 F20110318_AABRUK woodling_k_Page_107thm.jpg
bb9473e4750aa18c7bf42e4f65c3a90a
35da21bad47fbda75785daaf6a33ec85970f830d
1924 F20110318_AABRTV woodling_k_Page_088.txt
cf790c6c9b296b68e65aa8004c3071aa
ffe588731a52cb8b58a7ba89b0268eef596ca61d
646 F20110318_AABSAE woodling_k_Page_002thm.jpg
ca038e3fc5c98d7413e02dfe0c5b80d2
0efe12d43f1065813fcdf4ac40b601cf4bb98b5d
25411 F20110318_AABTCT woodling_k_Page_093.QC.jpg
498801e4bf33449dc93df4553db5fffe
1a730a1d711a58fa0ebcfa92a475784377597e7f
7563 F20110318_AABSYB woodling_k_Page_064thm.jpg
061b79d46567aec90f210ed61eefc95d
0d192b6ea8e39b5f81607934c14f2be35a755579
1993 F20110318_AABSXN woodling_k_Page_072.txt
949341dd012b61c1005934901be45c3b
16d0a3dc271e30d292f1870150c0323128fbadcf
32555 F20110318_AABSWY woodling_k_Page_066.QC.jpg
a431f5837e30bf65df40319842b8a4cb
b3ee0dbbc8a1af26712730da67c6c14d448431f2
35130 F20110318_AABRUL woodling_k_Page_108.QC.jpg
9af770a554134a69f9fef3cf9d953919
ab2ab44fbb338ae500f5d70a8e6c62d2ebd5007a
32914 F20110318_AABRTW woodling_k_Page_102.QC.jpg
65dd3669fcb9c69923a8f3b4fed24908
a30348a488b84911cd92e9631e6418d235ee544a
6552 F20110318_AABSAF woodling_k_Page_061thm.jpg
94d80df86585d17bf8a9921885f0c010
fe8d8ceead798f81b88208b9fa9f12d6bc40ab4d
29291 F20110318_AABTCU woodling_k_Page_105.QC.jpg
f206c3cf4c991f625c1394fb378ee49a
5990dee212eba367b8032404cebc9dc7c8072278
5997 F20110318_AABSYC woodling_k_Page_002.jp2
aec77a51494467a352021a7a81bc8be9
67189edcb201a098f59387e7fc404a40985bd284
27666 F20110318_AABSXO woodling_k_Page_052.QC.jpg
2a0d64df98a15b599862e523d791b0c9
c5c1afc28793dc350f4f14fced56bbf086de5582
2072 F20110318_AABSWZ woodling_k_Page_018.txt
2a39d62b4c78ac9de41bc03321a6a2d9
51aa65ad433d6d47675c53c8078e83d4bb55f5f9
F20110318_AABRUM woodling_k_Page_080.tif
870c26af9f2ec6178a8abd2b3ed7a9f3
92eeb6f8c075ceb3b2a062a8520ce45f410372a9
5988 F20110318_AABRTX woodling_k_Page_092thm.jpg
f4445e7d3ffa177052be71a3a53fb738
6f5dd93ab0d6967151783adff615740e1d09a713
105199 F20110318_AABSAG woodling_k_Page_031.jpg
9bba2935ebbe1f516d57a18228db81b2
d9effbd9801e50e0827e033ab2e8a09bba5a7c6f
100990 F20110318_AABRVA woodling_k_Page_133.jpg
2423135c0ae826472090e44eb82f493a
6b259a3f65a20e1e1a0b024c50a3e4fccf67dd30
6189 F20110318_AABTCV woodling_k_Page_006thm.jpg
ea6a245d672134ec7a9ee1531331618f
75a94c10c3b28a8c063f59d3b245c3f4d9ffe0aa
35181 F20110318_AABSYD woodling_k_Page_095.pro
56cb7bcc011b679db282f4d16d89f987
942e0cec15327e2d7e9562d650c352776568d320
942979 F20110318_AABSXP woodling_k_Page_127.jp2
11c3158b5b7343266649b616a0fbd2b9
87547ba4d4f75fc18d0d244d04fa360cee1b6cef
7120 F20110318_AABRTY woodling_k_Page_042thm.jpg
fa79571563d260b8793fbdf436513ada
257fd9c3b6b63f118080ead2662bd307b323272e
F20110318_AABSAH woodling_k_Page_036.tif
a81fcb68b1c16e2e5d7f5526bea83cef
a72dd82fe8785c470d873c9d5ec2677b76b24e34
73458 F20110318_AABRVB woodling_k_Page_083.jpg
df47609644395703be9577483b81fe2a
a4d29c11370973a4ae906bf1a78bbfef88dd8637
480 F20110318_AABRUN woodling_k_Page_003thm.jpg
dc86e28fa6b8c1dc5c39ffff7989e3d5
d85add45bdff9e3c1ae510b23ccc34b49f10e906
8524 F20110318_AABTCW woodling_k_Page_044thm.jpg
a17c53fdb273415299e7e1da5d120404
f6e256cad81590ab452074d582f0c5bedb8d04f2
1816 F20110318_AABSYE woodling_k_Page_136.txt
5208e8d768520b549f749c845f5098a0
e3bb6cefbe77dfcdaa222b766430e3daa3d4b0cd
102446 F20110318_AABSXQ woodling_k_Page_072.jpg
7e6e18d833aeacf75f8a7de5d4f12005
376e37a40897ef9b8cc71d35b08ce820284436f8
1572 F20110318_AABRTZ woodling_k_Page_120.txt
d53a0ab905fe13924b1bc9390a4dfc55
4946102b82bb1ba6cc487e6488d6fd1d205034e9
31792 F20110318_AABSAI woodling_k_Page_124.QC.jpg
cd6dd23832fb62480b8672ba6a8213ae
4b28ae474a10de801a81b56fbb79df286d35dd49
93396 F20110318_AABRVC woodling_k_Page_104.jpg
44045157e1271c9e76d531290eac2104
6b6d9a04a904da1fb251de2e3dfebf0b68b0b54d
6192 F20110318_AABRUO woodling_k_Page_129thm.jpg
93536baf537752f248de930393574ba8
a7319bcdf2aa72c9791dd124a542e59ba9f4906e
8560 F20110318_AABTCX woodling_k_Page_087thm.jpg
707f9ee1d7dff78d2d20b94b0e6b75bd
599993f7c37ac4ce37938476a0614396061467f0
99986 F20110318_AABSYF woodling_k_Page_030.jpg
f2e528a0796efeed64d0b5d110ce19d3
d992c68159982a067d45dbebf9e9da4f886e1fa4
90471 F20110318_AABSXR woodling_k_Page_127.jpg
050fc33ac717b878ca51a8456c9d7662
75ddcbe34e61924cb7ec1976244da091814bdcb1
82330 F20110318_AABSAJ woodling_k_Page_098.jpg
1aebdb16e57f117075edbc8c9b6149cd
c81fe8651e2ae323f3f0424ce7426e3dc0a6f080
102118 F20110318_AABRVD woodling_k_Page_134.jpg
2054e7601f1224c3fc233bac3a9b8ab0
bbd8a24190b272f1ed0cc7e1ea21951cd804e129
8254 F20110318_AABRUP woodling_k_Page_028thm.jpg
39c21a3352c0afcf518e02d4a8894bf7
2bc1caea31846eada531f67fde824f35b0a55935
7400 F20110318_AABTCY woodling_k_Page_131thm.jpg
eb0cdc68fa07f09b558438b4d5b48f02
ceb0267fba93708573d91bf3a1aa9e1b260565dc
112816 F20110318_AABSYG woodling_k_Page_108.jp2
f2ff3a33475b4315507958c435b83491
9e61b7bbc2eb0ada40c1ba905a9a16e103f8caec
16798 F20110318_AABSXS woodling_k_Page_009.jpg
ccb1195eb8376e5fd57af9b2f27f75e2
661629e3207e0aa6ae7d91aab446e0d3067e7c96
961887 F20110318_AABSAK woodling_k_Page_070.jp2
41759d52b48c6817462d2f5cd4e857ba
9f128e1624223a15c26c887172dafe269d528648
8229 F20110318_AABRVE woodling_k_Page_102thm.jpg
694d503870eff0d40b2a88aa1944952c
5ed936e2775b14de2aac7d218cf1970236c7759a
F20110318_AABRUQ woodling_k_Page_143.tif
5af692a0bd853613ffd933129532f0b7
bea43b7264066e74c85279d486976363a9d2d387
8285 F20110318_AABTCZ woodling_k_Page_132thm.jpg
999134b38dde8b5114881226dba51951
e2ba93a9a5183bf81b3740555ff51d512a156cca
1729 F20110318_AABSYH woodling_k_Page_050.txt
ae9a84cbea33e4c6f325489e84448ed5
c60392a3782ce80c8cb6674bb78eab0919c22da8
8057 F20110318_AABSXT woodling_k_Page_127thm.jpg
b4a1e3f007cc7474e9de431b18d3d53f
2a048015de70d02d3c92e6bcdebfcddc5b722a5a
1714 F20110318_AABSAL woodling_k_Page_051.txt
8e169172d325f44383ee9ef38b87c43f
1fb3beaa212c3e3e68bd95dbef7f86f2265dfdea
F20110318_AABRVF woodling_k_Page_013.tif
d0228523062e1cef9645c2cd814cb261
d229d3c9c49dfa0374be4ff7936981f857043594
5195 F20110318_AABRUR woodling_k_Page_130thm.jpg
63716cdbcc9a676fa2eff03e7c9b087c
25bbf74f1e38038ad62bdf45c73d33d0d39bf0b5
314 F20110318_AABSYI woodling_k_Page_009.txt
39b3555d34c6ba3ffd93c18acf9ea004
5611c7ae808537bb0d9969ad2f110e741cb2541b
7217 F20110318_AABSXU woodling_k_Page_100thm.jpg
e2d5dda33eb63ffd602c855637465280
4ba16b6330e64afecedee2c29fbdee9f2b6b90c8
94130 F20110318_AABSBA woodling_k_Page_114.jpg
a0f1952525453b160288d322b2778747
cd60b7f5883673571db02e52c42e5422cfd337f0
F20110318_AABSAM woodling_k_Page_018.jp2
d57baa530c1c33c89addc43eb28ced3c
d081bb6ffd0756c4c8198821e89fe6cfcea0beff
1051984 F20110318_AABRVG woodling_k_Page_106.jp2
17a1dcbf45bc8f97538f571287bcfde0
4d167601d8f8774cdb09ce4b66f7a71a08997227
1986 F20110318_AABRUS woodling_k_Page_031.txt
eeca9e0942ab896d8088f787f541f770
e3101cfffd97b3093b8c388b84fa33dd566ea2e3
1590 F20110318_AABSYJ woodling_k_Page_008thm.jpg
cbce80bab47397266660b70dd69635fa
9922f6e4a92f8ead5de3f633f242e602e370076c
F20110318_AABSBB woodling_k_Page_071.tif
0cf84a124a5803948e1d5625c5a6519d
08eb292bc1dda13b18abb60a8f576380fdc52f35
25194 F20110318_AABSAN woodling_k_Page_081.QC.jpg
27e348cbb4ce8c2688033d0a4cfa70e6
b58e59e7c12e250dc4c2af0907b2bacef55218be
42661 F20110318_AABRVH woodling_k_Page_104.pro
7b45f45b6b6d71e0cbb1af5f782ba1a1
fa3e99cdf1b2ea66200768bd9b41cb2e1fe3f329
44551 F20110318_AABSYK woodling_k_Page_136.pro
6f833ede91aa50492c7d3594ffe63b16
a660ca489309bb9df4a7938bebfbded92e97e43b
F20110318_AABSXV woodling_k_Page_020.tif
411abd8d4565e9d4df2a983a10b7e64b
f6721af35dfb003caba18c68ab997f6bc3b34f7a
49784 F20110318_AABSBC woodling_k_Page_134.pro
1a4ce59ba18a4bb2bc179457894f9494
3e098988019f3d849e79f2d6a41513cc79ee5583
772114 F20110318_AABSAO woodling_k_Page_076.jp2
a046b02dc65e878ae41f985d698d143d
0f6b8cd4e55f3dc063a1070105651f02c562f5df
50395 F20110318_AABRVI woodling_k_Page_069.pro
da15ef2a38ffb50d5bbc67e0377cf0bc
0ff0a2f92b3b72be56acce7776c519b03df14811
6829 F20110318_AABRUT woodling_k_Page_081thm.jpg
ac31daf5f9fb3361a328d267472868f6
5126446bcd40d3e3c7213f941891a7b30168cca3
112414 F20110318_AABSYL woodling_k_Page_023.jp2
a948552ac9395a2721ce80ddd2ac0a24
ca33cd39aac182ce4065994155771ede9bcf5d6a
92357 F20110318_AABSXW woodling_k_Page_105.jpg
21e600bcf4458a41a0c0b7f533ec369c
d18617e2c568061a899f7a138ffd805078864ca9
F20110318_AABSBD woodling_k_Page_073.tif
23c35f9d964cbab2bb3655893d8f0119
a1722d3e201a9458f1832512a872fd9dbfb98c24
8025 F20110318_AABSAP woodling_k_Page_141thm.jpg
0ef41ad071c4a4f89637dad75c31eaaf
2914ec9d3cf41ae027acd5821d0b61c55ce8eafd
1799 F20110318_AABRVJ woodling_k_Page_033.txt
fac384c30b7bf7face499752c992b593
99f781ab676d0d348908876b08588c0fd1581ed7
48566 F20110318_AABRUU woodling_k_Page_137.pro
e102469199ce24be9e1abe3323f78a3f
89d0a7864fd61a64b8fb5afb9807002261068e84
F20110318_AABSZA woodling_k_Page_026.tif
afb61c6b35b755a41e3cf964943a2f1a
079e89122a44e744d0efe7f94b83983fd2a51c55
F20110318_AABSYM woodling_k_Page_070.tif
bb80a51d7844aa54daeb6960cce2feff
9fb182c8e0d44448af8fba601fd7998f5bec9bdc
F20110318_AABSXX woodling_k_Page_119.tif
4844bd7a16e5361f4d393869687c9965
8a89088303276cf622af87547df3508b254e6957
F20110318_AABSBE woodling_k_Page_087.tif
2e62cd3e92af8c0c0e71aef1cd6b11d0
78f6417cda2873fd5149b01e1eb7c32c7694fb04
41710 F20110318_AABSAQ woodling_k_Page_042.pro
4ae7314839baf5274472843c8e117993
71117ac78dfefb2c0e372192e1a07353ca39bebe
35299 F20110318_AABRVK woodling_k_Page_101.QC.jpg
a9bd5794877c52d032867e884338c553
6351f5cf0480ee30a501b1e2fd093210265d279d
88179 F20110318_AABRUV woodling_k_Page_014.jp2
572acdcc8e97f968b8b32ee514bca418
02ced336b36687bca07f0f1cf31ba8d225eb017c
F20110318_AABSZB woodling_k_Page_030.tif
98d9adf9fe4867653c8a5b709cde2537
e238ed567ef2584d1af5316cb21333fec473d9be
114966 F20110318_AABSYN woodling_k_Page_087.jp2
da0dc826312b0808e79d559f6a46376b
f808558ad4b59fd063276fe7cdd41fb07aedb30b
102011 F20110318_AABSXY woodling_k_Page_107.jpg
7ef13650c69967daecdfb507a25d7332
0bb9afba4ff1c62090dc25cf8b6151bdd58ae301
F20110318_AABSBF woodling_k_Page_135.tif
1407ff87d5fd830950c6255cb4f0a5d4
650dfcbdfd24f1494cbb64ff0e66cc0b94f7044f
1403 F20110318_AABSAR woodling_k_Page_046.txt
3f37cb5011ff5a2329777f442a13f813
994dbe039dcf311ac3d77537fce12bfc09830dc6
91346 F20110318_AABRVL woodling_k_Page_068.jpg
002c4ee5da34adb45c28d3cac535c46e
f3afe2757289f1aa45ee1d0c976eb65f2fbea2ef
4074 F20110318_AABRUW woodling_k_Page_003.jpg
068fbf070d7bb39044b0a2deabffbc23
757f26b72513e2a45e29da73e8da1bd91804c5ef
F20110318_AABSZC woodling_k_Page_039.tif
afa9dcb38b200ede736ecdd5a08ba935
9fc23caeb87e2851db858192f35f3d23f619203c
41583 F20110318_AABSYO woodling_k_Page_112.pro
6ce8fd1190c21c15b603397b5245e854
ca831e1690097bff8daa286d10e96f12ed5b5968
107835 F20110318_AABSXZ woodling_k_Page_010.jpg
f18b0d98d8a84522f5d31b63734f7e0c
6b64ccd657c2ef5e737a1cde12884a57c1023d57
96590 F20110318_AABSBG woodling_k_Page_064.jp2
2e9b4972ec5fcc2c4c0f4a3577e8bc25
1c06399e36ebc7ae354290d2a84626f7ffe8f5f1
7891 F20110318_AABRWA woodling_k_Page_112thm.jpg
cad98137153a47609856983807d0e281
8a544a1743724773670b86a947c0a5a9b8113ae2
30810 F20110318_AABSAS woodling_k_Page_064.QC.jpg
585fd10d06330b47ffb94c35f86bec44
12ad7fd61fc44072bfa24581831cf0add4eb53ae
F20110318_AABRVM woodling_k_Page_095.tif
069ff893f19a1fdcb3ae43c1680b14f4
81a1e3c51a64c92a9dd1e68862bb61591ccbc323
F20110318_AABRUX woodling_k_Page_063.tif
aa1ad8460536746c8115f2c779e7c2e2
560521d4e85df1e5612aee5b5bbaf37cc7417c34
F20110318_AABSZD woodling_k_Page_041.tif
2cadc90c453f7b3600d89d41705f4623
873005d7970f66a516f2b468f1055f45cdb94734
51910 F20110318_AABSYP woodling_k_Page_142.pro
35d094bad52a8ebba406cdd348358def
e54bccf1b135d81bbfcaf01dc4382f04fb5a94d5
92483 F20110318_AABSBH woodling_k_Page_016.jp2
6d5986bbdc32d2cd4a7889d4e498806a
aa3c485e38adc2fd1bae56a2f0424a8855f34ac6
9337 F20110318_AABRWB woodling_k_Page_001.QC.jpg
5cae34ed24098cd52d050074906ec4b4
cf05bea0260e2c7cf58016e468ec15b238ae2c7f
101464 F20110318_AABSAT woodling_k_Page_137.jpg
ea61b9d2614c78616e72eed1adaf3066
116807a62fc95cc9d7165ff989b36fcf520629f7
29665 F20110318_AABRVN woodling_k_Page_112.QC.jpg
c3a06713683f9130fe7bf634bddfc33f
143e7930babc15f15d15c0002065f4735a404205
49707 F20110318_AABRUY woodling_k_Page_101.pro
a4ffc2aac95680ae729875209cb2e62a
bcd58ba28449fd01c5c8824af42f165c689f1848
F20110318_AABSZE woodling_k_Page_047.tif
76200b2648489f8ac58360a7730c4c04
b4376fbd199cb7d611ae3d6e771cae95707acc7f
248357 F20110318_AABSYQ woodling_k_Page_008.jp2
7832df360af95ef7bfb0220e5a87fc46
8b5a0eeb8f2ded56499595f80da40dccd736cb8c
29915 F20110318_AABSBI woodling_k_Page_052.pro
ab22d21e6590037dcb113f806b38037b
b91f5da848f2be69690b5a6d41d3207a9ea20c12
99371 F20110318_AABRWC woodling_k_Page_119.jpg
bf9032acd8b8008cde6ed60027d9412f
43e669e4649df07f9428fd6c38300ff8c40868bf
92947 F20110318_AABSAU woodling_k_Page_112.jpg
dd2f7ae407a9b3ee202701310a83a9a7
4cce2d6899c5257990286b1f3304579567bd243d
1948 F20110318_AABRVO woodling_k_Page_121.txt
4fce5ae9f6a5a9b568a21252a4129d42
cef9fe8fda10c7111ddcade630d6283c95bb5854
F20110318_AABRUZ woodling_k_Page_029.tif
be482fbcd4d8ca59e37a6378671c368a
401861ae92be807f723eff626c900956e666c950
F20110318_AABSZF woodling_k_Page_051.tif
adcc77e91f34ab4ca497e4ed637ec931
122e110fa4bd4090723cca79cb99fbffcab8635a
108439 F20110318_AABSYR woodling_k_Page_028.jp2
5924c5aaa4f66b989043300eabda2996
458b1bdd1d7da67630a2a62c750a32abb140b94e
F20110318_AABSBJ woodling_k_Page_053.tif
711b2335c08f447943ac41f3a24a457e
40efdbec97720721ca3669449f456a7b967be79a
102191 F20110318_AABRWD woodling_k_Page_085.jpg
686c3dbcd3cf788d2a5befec25b8d63d
04866b229f9beec89f35368d3dafa156627f162f
1051939 F20110318_AABSAV woodling_k_Page_040.jp2
6c4a846b2993f47f605e6d14709dd3df
8654f4f640fc8a638e458963318aef5b4ce79781
F20110318_AABRVP woodling_k_Page_016.tif
d177f673fb23b798d5c1c210f4e94d5c
1d51ad50484c26aea0aa2e3859fc3664875a4f34
8423998 F20110318_AABSZG woodling_k_Page_054.tif
cc1b34bddcba3761cf69cd9cf6702075
9f73f2b507e0dd0f8b2ef4cddb1e91fb2e658067
8187 F20110318_AABSYS woodling_k_Page_105thm.jpg
793156ecce6771e098dbe57707f01960
b03fdd4f2c3eeb9f0ee462fe9f9dd53bb49cf7b6
19159 F20110318_AABRWE woodling_k_Page_067.QC.jpg
7cf9d0227f8f5c77db55bd02f5a2f246
74c0079a8a02b7928fbd5214850747fe24a2a72d
47797 F20110318_AABSAW woodling_k_Page_073.pro
d1008f96711cfd7a760a593dd31859b5
1346d423c63eef432f4d0877d251063055a98d9f
F20110318_AABRVQ woodling_k_Page_118.tif
377ebf72241b2a11b6bfb3ad2d7eb7c9
a28f0b63e8f4df29f6ee3cc9902d79b4d767bd8c
78862 F20110318_AABSBK woodling_k_Page_083.jp2
80ba866b9791295e3a8cca6212335f82
b4fe49f340d2575cc1d71f0a2eaab8f3acb77910
F20110318_AABSZH woodling_k_Page_056.tif
70c75bb12a282e72b0718c2affe9220b
47258445203401aeaf63939877fcc5c7d1c02ebf
172878 F20110318_AABSYT UFE0012801_00001.mets
843a9a208a8064c0bfff323835d4ae0e
c91a0093db8fb3ec695ded6006a0d6b683739cf9
8064 F20110318_AABRWF woodling_k_Page_060thm.jpg
bb5ddae37c00309026ff303a58eabbcf
4f3434261eced030ac97307e6da4e5cd1640c6d9
33927 F20110318_AABSAX woodling_k_Page_039.QC.jpg
a1800592dce123a5aa95e60e69033a5c
5dfb9fd1a40d407b9ab0dea50e09b247d9d3a622
42749 F20110318_AABRVR woodling_k_Page_070.pro
a0253ff57f400d13f9c4a26f070ee1a8
668df2a4cf4f5501dcf522be60c724c50e68141d
750745 F20110318_AABSBL woodling_k_Page_061.jp2
2007a996c9d59f36bf3cb9644d6ecf46
21a8e310d79aeac23a9a2afd786c45615af607a5
F20110318_AABSZI woodling_k_Page_061.tif
e223b6a4d2bd1f7387ab469276fa0185
03c77e8afd0220db1f5c8d16c4913385c67a3238
41026 F20110318_AABRWG woodling_k_Page_048.pro
c75f4464536e429cbb61fc27a4d87260
02a80104ca307d9eb6ac1c32119e81cf60da27a2
F20110318_AABSAY woodling_k_Page_078.txt
d9f7e497a55af04b6a193273c3669d26
492dff06dc43351d3714954dfb71c4ffab6ef793
2062 F20110318_AABRVS woodling_k_Page_065.txt
0f5a7ea5f6b90181a16a381f0b564b69
fbe21a55d0dd4d571cc3358a6d4c6874afd15edd
97567 F20110318_AABSCA woodling_k_Page_140.jpg
da1a8234e0a29ad51c729719fb8f91f0
de6b748e9b803709da33246a64b1d27afe655af4
36904 F20110318_AABSBM woodling_k_Page_093.pro
8944bd373a0bef879de38fe5a57aca00
77b9365538b9714233b891f65c810fb28422032a
F20110318_AABSZJ woodling_k_Page_062.tif
a1fdbb7d28e71e65c4b586f6ffdb7754
97c22279c7ffe240b4a9858e6057aaf5aad0a8e4
49282 F20110318_AABRWH woodling_k_Page_029.pro
e9f84887618135442f48f34089514a47
031f936d5f49bb052bec2cc1199ff86e59778484
6158 F20110318_AABSAZ woodling_k_Page_051thm.jpg
794986e9990aac1a3986ed5a7fdd83ff
4faeb20b1d4e277980125d10512388c3c8a43df3
1491 F20110318_AABRVT woodling_k_Page_098.txt
b7d8bce3d3d0c4bebb59db0178e25cba
51925907af1780f44bc7ba7974214be70ea24a99
48656 F20110318_AABSCB woodling_k_Page_030.pro
a1e3e103703272e7752d5949e36f6356
7efc69aabc928f0e9f2a313183b9f2165789b2b6
78369 F20110318_AABSBN woodling_k_Page_094.jpg
55c923bfdb6508cbb7ac2884262e3444
f960e367010fb288df4102bb64ef92ffd241cb4f
F20110318_AABSZK woodling_k_Page_092.tif
1503ea09725eb3dd2afd74809b42b9c6
78d712f96091dc83f6bd96e59cd948dd209f866a
47594 F20110318_AABRWI woodling_k_Page_063.pro
dedc34c97ee4f68291328d7fe94a5525
bcd5479422bc626f8d528c07a9fe9c4c789d796d
8130 F20110318_AABSCC woodling_k_Page_047thm.jpg
076612e984eefd70ddda990b6b89c2cd
de83dffe5591e9cac6687d8ac61fdcb51588c7a4
1008777 F20110318_AABSBO woodling_k_Page_090.jp2
2bfbcff3f812f029a2706ed5d1827d64
568507fbf3a16d3928b9599c6b544fe9de3b53d2
F20110318_AABSZL woodling_k_Page_098.tif
c7ebe89c9eb14e9195844eecfe7bb731
2a3ac175414c05fb201a1fa1b8c8b2059186e58d
F20110318_AABSYW woodling_k_Page_004.tif
66683bf182cb2fbc7f252806b2bca354
6954acd77cb5dbc7beb874085bad24746d3cab78
39643 F20110318_AABRWJ woodling_k_Page_012.QC.jpg
0ddb80fde5432ed92c90652cae8fa02a
0d84ce643afefd543dd07afc92d44acb7819851c
F20110318_AABRVU woodling_k_Page_113.tif
e78f6b1a63091d9270bf54da4f5fa087
7e68c23bc371c0caeb471439ce3ec70101724db6
33639 F20110318_AABSCD woodling_k_Page_094.pro
a79c2db2fcd701cecfc8b8c0b66f9498
695e246c9700bdddcc8223739b125fd7bd8b1211
72609 F20110318_AABSBP woodling_k_Page_123.jpg
f4f1d7928ab08796cf870047d9bec6b9
34b6db216b4830cc5c7b4a11e1c01b01db73489d
F20110318_AABSZM woodling_k_Page_108.tif
ee5e1a02b7c371c24510d514fc36e566
322d33cef086107eee30194dc9ab81dc7652a4de
F20110318_AABSYX woodling_k_Page_005.tif
2c4f604df1dcd5bf30cfdcb2318867fb
64828bb7a23b71943e3311c7c997c50cc6951cc6
28280 F20110318_AABRWK woodling_k_Page_089.QC.jpg
e05999adb9ebf6c950714042339e3eac
871acee716d6af9ad2eee16eed5f983ce61d9b00
75548 F20110318_AABRVV woodling_k_Page_077.jpg
3154bae107a6d91c282e32370bd2c1c7
165c76e006da6d5786ea887ce3dca7ec9b0e4e83
91240 F20110318_AABSCE woodling_k_Page_004.jp2
a164e0e8721ea495f4c2fda18cff4c60
fb11364ba90edea7834b623b7499c9ac3d110bbf
105115 F20110318_AABSBQ woodling_k_Page_045.jpg
92d9aba815a813438116b392a55d03bc
30fb618150b090eacb1165bbf3855d72212469f9
F20110318_AABSZN woodling_k_Page_112.tif
2584565c2f20773635075b0f5cfc08ea
1793ee57c30fb32d58ffd0f7abf251f7b4c194d9
F20110318_AABSYY woodling_k_Page_010.tif
4b5bf0e5b31190c3ccad795b94388a69
7b562b5556bc62bf5a20f1bc8d859ccbd53e0b11
F20110318_AABRWL woodling_k_Page_086.tif
e971dc9d2e57ef75c43215bfa553fa50
a3e19180630f054b3e7ca53c581200f017f29621
103334 F20110318_AABRVW woodling_k_Page_020.jpg
f0fe30ec5736dcfb48f29fca8ce48cad
a96b1a8fcac9259db460383554f58635362a00c3
7905 F20110318_AABSCF woodling_k_Page_136thm.jpg
c00945b89f15548ba724373a8c1767f3
966daf2693999d5cc8e10c0136e40145b6180de6
34004 F20110318_AABSBR woodling_k_Page_086.QC.jpg
f9160116a6e100c550f46d0fdb26cfa2
bc89c1c6207b61c9112336a7074aa1a25ca50e29
F20110318_AABSZO woodling_k_Page_117.tif
6e12873497493e79915754b4f48ec0d5
1342b9d1c796a37a72532ad3d62e114f2d6fbe94
F20110318_AABSYZ woodling_k_Page_023.tif
557ac2dce75ed9669a3c54bb810208c9
f8b0192f9fc8b2de8706e8a2b534415be5056d1b
33007 F20110318_AABRWM woodling_k_Page_118.QC.jpg
fef68aa186a37171129689caee6dd263
849f46473132c1b6d9ae098d1a34c7f9a9d2e0c1
101949 F20110318_AABRVX woodling_k_Page_028.jpg
3f618c44a70d3fdc3d54c5e4981f5f6e
2ddece1da40ae1464a83587b3fcdeee4481591b0
F20110318_AABSCG woodling_k_Page_016.txt
fc42ba925e1556e62cf28a2c271203ba
edf190692741f793e58043f76dd5d6b9cd96a8d5
74379 F20110318_AABRXA woodling_k_Page_061.jpg
f5aac8cf98e6de21e3617e7053aca444
5593d73f2ee6735cf5ba4b779c67e28a8ef71843
45525 F20110318_AABSBS woodling_k_Page_126.pro
c01f9320eb9396c42569e34ca4d84d48
88f4c131ffd78d77e8344c4e0d136dfb3c8d0ea7
F20110318_AABSZP woodling_k_Page_120.tif
419ac7c06ccfdf74b009bd8e97aad240
6868fe3475c39f6744a23a724f688ca8b3d44e2a
8041 F20110318_AABRWN woodling_k_Page_070thm.jpg
017bd4e8874d0f3203b49e51daccfff9
068eb298e07fe30a4b05cae117e596ebb973a2af
7774 F20110318_AABRVY woodling_k_Page_078thm.jpg
8ff49a6bc7e6f48cda4879b7f37c0910
92889e481d6d7071c588ef9f55d199bcfcc59495
F20110318_AABSCH woodling_k_Page_079.tif
8ff29aa359167ce0537d3df23f594188
d88da4919ae4f9cabc671d36a0dd804966e94f01
81615 F20110318_AABRXB woodling_k_Page_048.jpg
cc878416474d380617966638fb9177cc
15283128e8e3db98b9bcb850a73eac164ebf868b
7983 F20110318_AABSBT woodling_k_Page_104thm.jpg
e105cf63530aee10eba7feb95ffe2c26
5309219d0eb82886abbdf40fa778eb4406306453
F20110318_AABSZQ woodling_k_Page_124.tif
77eff1d766b14020e3bbd73700e8bb93
677ccacabfb62bedbd80595a8011e068a41878b9
6707 F20110318_AABRWO woodling_k_Page_014thm.jpg
2c40680b81c89ce9afe54d36a52bb3ae
e3b1d8e277567bf14156f6f21aaea9aa301c0418
36613 F20110318_AABRVZ woodling_k_Page_040.QC.jpg
cfc960fed2c3ac7d1cb7ddc179a8e7d2
a41975a5e495e62411b63eb45ef0ef28181a86b8
826322 F20110318_AABSCI woodling_k_Page_110.jp2
51f0d7fb765961a692e2e4c8f4564e2e
84ea1abe2ccec1ffbed06c9778ea1c740344572b
F20110318_AABRXC woodling_k_Page_031.tif
cbbe19e68e9ef85fc4867de2e048fee6
c187e854ca7d216e13f43d16c054254710f5bec7
27748 F20110318_AABSBU woodling_k_Page_016.QC.jpg
a2a1743fd241c7e3051760c382eda815
5c2d61dd30e7ee15a4d83ca3ea3d1054dd0fdf6d
F20110318_AABSZR woodling_k_Page_142.tif
360403928bddd0ee585fb85c1061e4d7
3555638da3a2a847e16d627110681b30db0c6329
911638 F20110318_AABRWP woodling_k_Page_145.jp2
0118b3a596e61d08cee7aa5e576b13a2
90955a4bc3a832de7b3965f1a757749159cae618
106404 F20110318_AABSCJ woodling_k_Page_142.jpg
55fd673916400b070f1593ee0cb4e0cf
fb518495253bb07e5361f82d0e4dfe2e5ecc8ede
75829 F20110318_AABRXD woodling_k_Page_080.jpg
a7abac2a7201149f71dca4edda112553
7785761143f829b985f914be4e24f63ca199acea
1861 F20110318_AABSBV woodling_k_Page_140.txt
c531d76a4086d1dc4676ce90d834184f
7c96ffcea8fd98b737af37d4a6b5b6f5ecb15ab4
2049 F20110318_AABSZS woodling_k_Page_020.txt
e4922bbfc986316faba5cf7a7cd2442d
0f3bbdb59c266d635628fc6b1bee62c59cd6b19a
7103 F20110318_AABRWQ woodling_k_Page_033thm.jpg
68358546a696437e9c88ece62a507160
f706b040a91546cdda1e437592773193e0811a0c
4294 F20110318_AABSCK woodling_k_Page_005.QC.jpg
275ede52d30fb5d74047f2c4e9089d39
be84b24af7e7d78a8820ca0291550d3aa89f09bd
5984 F20110318_AABRXE woodling_k_Page_082thm.jpg
8859d611bbc398f9b3d3e864191c2d63
06dfa49eafaf4ac7073b9802457bd3395ac66383
F20110318_AABSBW woodling_k_Page_082.tif
1288e69f1a670c7a98b43b985125cd4e
d6a8e00436ee20a3ab517286e251eeed14236536
F20110318_AABSZT woodling_k_Page_028.txt
69edc1082d97cc4a38cb2b91e1c0705c
adacbfec02430215c87f0fae88f7e204e2687be3
1762 F20110318_AABRWR woodling_k_Page_014.txt
8aa21a62b521d4f64c157f8d8b0eb8f0
c5c0dfc45ac22ed7a42cea81b8c464d53d54098d
1982 F20110318_AABSDA woodling_k_Page_132.txt
033ae9ee8fdbbf9332fcb298d2c61dbc
00db6643e154742881e36c291740320c69f02080
8400 F20110318_AABSCL woodling_k_Page_054thm.jpg
c49447c88489a7bedf5be311d0c59afd
089a709baf962fb31d55dc48007de447445bbb42
F20110318_AABRXF woodling_k_Page_130.tif
ef59990c761c9f4df6e2c0a5876eb7c3
6ac0078b3e7fe4f73fa7627c6e89aa6427bea6b7
69593 F20110318_AABSBX woodling_k_Page_082.jpg
b04a51d11d0c85560ecb75d981ff8925
db08393c36c276cf10fa37ee0537cc4881d9baef
1916 F20110318_AABSZU woodling_k_Page_030.txt
f7e8c7e30f0066543716f245732d4231
4553b563dc098bd8cc80780e793d2e212088ae3c
1927 F20110318_AABRWS woodling_k_Page_113.txt
bf9f4b8d68e6ea71fcf806f27ebf5935
f2c29082abcd05461e2f9a495da63d6d2ee72fb4
22786 F20110318_AABSCM woodling_k_Page_076.QC.jpg
7dcecf3361666976889b4183a941ed1a
858061ea7743e702f6008168d38ee478be0644e3
109839 F20110318_AABRXG woodling_k_Page_132.jp2
7634f07b4cc658b7df4ff4c2748287ff
29eb431dbf3a16a0dc5325789ac260dec7ef57e7
23250 F20110318_AABSBY woodling_k_Page_061.QC.jpg
5c266d487482aacf0c617a0208f2724c
da9669aecda7cd2d36e5e85df380916bf4d51b34
F20110318_AABSZV woodling_k_Page_035.txt
0644f29ba9408c92f88fb3263336507f
570f4beafb6c14c144b7ec0acf7216c211013710
106911 F20110318_AABRWT woodling_k_Page_143.jpg
9213308210e425c6d865e08a59f9ce76
b4759f7613c013dce4ceb1a4045824a4795b8b46
F20110318_AABSDB woodling_k_Page_042.tif
732e7434bdc6347f486186edaf72cdab
b1edec244a92b0e696b48d10c5c7f44d5ee4440b
102460 F20110318_AABSCN woodling_k_Page_106.jpg
069d460029c2966599a47ced10dd50f2
cf659730817963cb9847b0344d61f79d1a05ecff
42948 F20110318_AABRXH woodling_k_Page_084.pro
87f68aef2988791f40c3048001d1a06c
603429e00c14c7567121a879109d71e8a059209b
36357 F20110318_AABSBZ woodling_k_Page_135.pro
52e46c9a0e927959b661d426ba304da2
b8d1e57a7a989bb5448639eb5e8c545a2d4d5ff4
1419 F20110318_AABSZW woodling_k_Page_041.txt
b6d525e8b5179cfd84f96fa463602f97
989920c952d5188ce212700a22b05f060753eaf0
6885 F20110318_AABRWU woodling_k_Page_016thm.jpg
0ec39ca816b5d68c5f684eac54a26a35
442e4b3a2858b602cbdd4c1932f5d72546633bcd
6853 F20110318_AABSDC woodling_k_Page_094thm.jpg
2d8f5e4e993307e730907b46765df954
5c3806a8a04ec28b355eae402f289943f2e3ae59
958792 F20110318_AABSCO woodling_k_Page_065.jp2
c3cd9a56b9bf6038741f64d26f45e9dc
168757fc7df934deee718ebc3c8add0b6a257d53
1839 F20110318_AABRXI woodling_k_Page_048.txt
feb6c46a463608f4a71753851cfec94f
256626946c4b86b3ba5d66f7d29fbddba3f56308
52334 F20110318_AABSDD woodling_k_Page_087.pro
664554078f36adc42d34ff2f1390afd6
b3f087608ce9179c057802a7130a253d0ece3361
97389 F20110318_AABSCP woodling_k_Page_113.jpg
6a9936655723fa8317519c1d2f92323a
a9ad5d0c324edb842c4f3490c6eb3ee889fd832c
95077 F20110318_AABRXJ woodling_k_Page_131.jpg
1e3dcfce5596f44c1b75c782d293c28c
d499c7c527f5b1aab3e9d740b94abc34ce52e991
2101 F20110318_AABSZX woodling_k_Page_044.txt
a008d0bcc68f7d982e2fb64551a55f0f
3c714201bf71f0608ebfff67bd665384feaf7d25
1436 F20110318_AABRWV woodling_k_Page_076.txt
a7aaf34d6840fb9e34a8ac5a2800bb61
cbedadd482fa876564b3c14c7a8b668ff511662d
6863 F20110318_AABSDE woodling_k_Page_057thm.jpg
bb41f1649478333269f6cafb06577bd5
7cbf4143c5154ac961159a69208deddb5d60db85
32733 F20110318_AABSCQ woodling_k_Page_098.pro
186abdd43acc58ff8993ca0e1d997ee6
a40d0a93c02c3c61e0a241d97b378329dff1df08
1540 F20110318_AABRXK woodling_k_Page_058.txt
0991cd7b49ede0e6db0190765658e807
ca7d866cef26578eb994715285e9cac986f72a7c
1819 F20110318_AABSZY woodling_k_Page_049.txt
49f53cc1ddbf97a250d6b59089d544b4
ac3202d03f771e7680f3f591d37e0b86a3055e66
6513 F20110318_AABRWW woodling_k_Page_099thm.jpg
830f8668a82f965241482f40ac4d8fd9
1254180d127bffec69008b79c74bf9c5518d26e7
1051969 F20110318_AABSDF woodling_k_Page_011.jp2
9c739db4a0fc7dc7cd2f98d162e48765
3c1c52c5d48ecf69be438cfdf9b0ae5f27cf788b
32902 F20110318_AABSCR woodling_k_Page_132.QC.jpg
d7a754ba29d5a8b0b1dfff3d4f0182a3
3ca8d4a4cd59f9d8c484c9d5161484e2ba24b9c4
36804 F20110318_AABRXL woodling_k_Page_053.pro
4c0de71af25bda00a1b316f927c8e4b9
6d98d6cf374b9f103b3d1bd33e05defc192411d3
1541 F20110318_AABSZZ woodling_k_Page_096.txt
d85ded1ea49bf2d93af00b90026ae43e
6e55f86542d7aeb4d233a9c3741dd6d5b779d529
33180 F20110318_AABRWX woodling_k_Page_028.QC.jpg
ee603f6b477733eb34b964c3c743c791
a3cb1f8878fb48263411cb107415f8345605a569
F20110318_AABSDG woodling_k_Page_129.tif
985589d19a10a6d589ee89f733b5b2dc
75ef36efe3487846a6c1c63a2adec2f1a55dfdbc
7763 F20110318_AABRYA woodling_k_Page_116thm.jpg
549ebacf3d35ad7b44a8bea981d11f71
bfc69a59f39fde8fca449ea909525921d05bf853
31775 F20110318_AABSCS woodling_k_Page_047.QC.jpg
024acf44c8681dffa43fbb832d1910d8
73c22272341f49fcb73f33af67a6c6a18155ff7f
7579 F20110318_AABRXM woodling_k_Page_065thm.jpg
9adc02b540a016d3d0c0136c66d9eeff
8a53975e610318f962f3efd6edd11dc64abff1c1
32888 F20110318_AABRWY woodling_k_Page_134.QC.jpg
62c544ed963d78e36eded9e0e914a4c7
6958e3cc980839c63b894e72ad235e008b8f9c21
80222 F20110318_AABSDH woodling_k_Page_093.jpg
e93c0f47b864bc25190c6ecd996377be
c13983faafa830c6c25e87da7bc977b4f1802c88
108017 F20110318_AABRYB woodling_k_Page_115.jp2
b53c6a4d99291ba3d7d276dc95049adb
eb84321d55618ed7ba53aa77466983d0769e35b4
31700 F20110318_AABSCT woodling_k_Page_113.QC.jpg
5f593a561202dbb620b8c366b6ae2fa6
f041dfb4906c83373aeedf05b6d806a35fdca265
30412 F20110318_AABRXN woodling_k_Page_125.QC.jpg
bbd7242b1f023cb89eb5d74fb903ca0f
33edacc70db9d345c1466b021b820fb267d5301f
8502 F20110318_AABRWZ woodling_k_Page_088thm.jpg
e8cd67e1eb93f2676dbd3cadf879e5cc
090356172c92cfd71626ddd98bf3a478e90fff5a
1823 F20110318_AABSDI woodling_k_Page_125.txt
bdc0face133db00c45a6147133a6121a
61caa790880ab535163b5f33cb7782e6520c2923
25963 F20110318_AABRYC woodling_k_Page_145.QC.jpg
822869974fb8f6166065702a23ac1354
c432455ac22c4f953af60d1b5ea4704953a6fbaf
7104 F20110318_AABSCU woodling_k_Page_004thm.jpg
635c2b18f032016b281ca29b0df37118
be5dde0ec756bb57a731a283c647ca274e377c5f
72796 F20110318_AABRXO woodling_k_Page_058.jpg
d5b923ab0005659afdf7695324d31a95
6045f9600a4f082db08806c8146353bb68376d4e
32918 F20110318_AABSDJ woodling_k_Page_032.QC.jpg
be0a39f89de2b6860301375fbc73612a
10d096cee62d51131cf3e2831e7cad66249e23b1
35823 F20110318_AABRYD woodling_k_Page_122.pro
5ecf7369965b0b00e3a776b4a5aaa265
2a9b4611b7cb5bc9e7b4e055de7a4e1e674c6cc8
7832 F20110318_AABSCV woodling_k_Page_029thm.jpg
176b274703dc042335d397f52035cff7
86bcfa0a310823e0223efe94726f10252906afd2
F20110318_AABRXP woodling_k_Page_122.tif
257551449cf02dbf39124154bd3477a2
ce11b1d0f9c01f0d8261f8f15a50a87d861e2e65
771 F20110318_AABSDK woodling_k_Page_013.txt
a30d74e4ef979b98e51f66f821dae7c7
0c6de7acb7ba906c6e85642a9c1dc556b9063ed2
8906 F20110318_AABRYE woodling_k_Page_008.pro
fd5cbf03ffce04ec660e603b32f39797
02341329fde93eab27a71c1711498c880a30f797
1547 F20110318_AABSCW woodling_k_Page_089.txt
2a2740e5777ec4538d8136109c889ee1
ac74e40c697bf6e50cb19e7e924de9cda9ab2365
963751 F20110318_AABRXQ woodling_k_Page_116.jp2
53fe00b88ca77ff254eab5bd9ccb3b51
b24857c47335852845a229e663c71d7c31772406
1944 F20110318_AABSDL woodling_k_Page_137.txt
a3dca69904bff7f83c55ad4df1e16faa
89b77f9b8b901ebc385b957c76122750334ca14b
107833 F20110318_AABRYF woodling_k_Page_069.jp2
8e8adb6b6ccdc5ed3ec107d7b71de602
9d5033c166cf78400457875804ff14f055a4aa78
8245 F20110318_AABSCX woodling_k_Page_128thm.jpg
17df180eb721229d103c2c123b162806
3128f437f03cba66a840e9ac8eee7bd876571708
8097 F20110318_AABRXR woodling_k_Page_066thm.jpg
5c08942d3fcacb66ffb62d73e73facb3
742ef97fcfa57d5ab27fca5a9b4ecc9a7b1e18fa
5880 F20110318_AABSEA woodling_k_Page_083thm.jpg
b0b5f900df977c1d0b90a4fbc2d8c020
76cc68ed83ace64dbf8c0deb5ba8446054e24d80
1732 F20110318_AABSDM woodling_k_Page_042.txt
2ee10c26cde6e833f397c50afa1a9f3d
618d5519b6afaed53b04503f1493c2228f9ce531
2005 F20110318_AABRYG woodling_k_Page_126.txt
38e136d138a649c258be9a2dcbd9d529
db1d90f091fb56497b5e7fa142080bd04b07c8fd
26265 F20110318_AABSCY woodling_k_Page_014.QC.jpg
9828ba8dec6b9da9ad73d996aeb9fc3e
d3bc504fd99cd82c9b115d833263557d3a7997c0
F20110318_AABRXS woodling_k_Page_138.tif
dab407fad28d56539b28cd85acf622a7
31062147c723fac51599aabee5b6f88c7e741212
31589 F20110318_AABSEB woodling_k_Page_001.jpg
8bf43587546e5aa9e50f029f256fa397
cb18400e92b75eed25e4109375e53259363c24ce
1770 F20110318_AABSDN woodling_k_Page_034.txt
d6795e2ec70f03ef1f53b19954d90c6e
00a5cc7c6588fdd971e863b4165cca1651922bd6
110459 F20110318_AABRYH woodling_k_Page_036.jp2
c137e7f0a51248dc0bf7df6d1199e667
739ec3fcaeb0d3457bd3ef9f1267c1a7f76298f7
1672 F20110318_AABSCZ woodling_k_Page_040.txt
235573e6ef233ad90d701eb060b4b58b
2d5d30fdea79152324c66bf6ba3e9de7ea11e918
75923 F20110318_AABRXT woodling_k_Page_135.jpg
e109ad5ad40c554f780cce5cdf29d29c
f6d6c883991354741bbb0c80daf7cd79c1c26648
3286 F20110318_AABSDO woodling_k_Page_013thm.jpg
b9f37fc8a42707ad5cb3ae89743ca189
d3335e8080b1de3e369031a15429a179543348bf
4951 F20110318_AABRYI woodling_k_Page_003.jp2
6c2698b9bc3182ef32412fbaab60d81d
178f75dda1f5c812df83ca4be7b9a629c5d25647
F20110318_AABRXU woodling_k_Page_126.tif
bdd7c7456517fc6cd02e48b955002ecd
2f3ff532a5f72c469221777cef438e6694749444
48700 F20110318_AABSEC woodling_k_Page_047.pro
5de33969b03c0c03444355a4f192575e
87f51f75d15d31dba27960966360e9f5ea0a179f
F20110318_AABSDP woodling_k_Page_017.tif
29dd98f90ab239d56fb0e8143714379e
3bd8b068f7fee37c539ad6d311d6adc9f8840d86
21977 F20110318_AABRYJ woodling_k_Page_051.QC.jpg
a2464623299268180f89ab8479574855
83167c47ff2ac883eb09145fba06fcc8cfe771d1
43560 F20110318_AABRXV woodling_k_Page_055.pro
df7427104ad8de76727c35d068d81c73
060dadc4d29f773d8b294fa2b5566e8708ac5d7f
78822 F20110318_AABSED woodling_k_Page_041.jp2
1057967cef9f735e5910c8754125db12
105c8033ed0204eff9d48b19fa3c6a81e28b740f
F20110318_AABSDQ woodling_k_Page_029.txt
22d98acc31615754c44c96ec750f4f17
2c0d3defe3e57fc980306f2671321c408153b568
106023 F20110318_AABRYK woodling_k_Page_108.jpg
b6165e3c2f9ba70cc2932253f2fe0f01
52c7489b4e352545fc3d1ad3a1696cc38e69f277
921515 F20110318_AABSEE woodling_k_Page_019.jp2
f5c7aafc020b41c85052cf11d5dc5b4f
5e31268731df7e22fbc731bd6228101fee25352d
27739 F20110318_AABSDR woodling_k_Page_042.QC.jpg
f102e66e713cb2b5a03f1fe8ceb03946
e15ecf924c62a917a3fcce9bf5cd43ad4ac41dd4
1918 F20110318_AABRYL woodling_k_Page_141.txt
d5a03491b78924a1609a69fa522c19db
6bf0d4424b5f1f6406d9926ab8cbd5802877ef58
8028 F20110318_AABRXW woodling_k_Page_017thm.jpg
5d84effc1bb405fe7a9c2f2833a432b6
de01e2443f84fe84d52949f76558bfcb9a182952
F20110318_AABSEF woodling_k_Page_103.tif
662d643ef503268d90b8e9c622f23b45
426c41a7d75fc8b14110ae7399349200da636e15
52621 F20110318_AABRZA woodling_k_Page_143.pro
e40b0ede64d32b4075352164af99c29d
a123c377216509d5879321f7be0415f1f7b869ee
2026 F20110318_AABSDS woodling_k_Page_071.txt
05f1d2da02d7bc9eadfa57dfef9c2c0d
b1ae1d411189fc9d5db9f98dbd0ee9e0b590dfa1
1706 F20110318_AABRYM woodling_k_Page_117.txt
a909e73f34512b52168e5a53fc75753b
e3849808924bcd97d340ea5f53af2dad1ab150f2
46057 F20110318_AABRXX woodling_k_Page_088.pro
4f2050be2ab28794c4e4e5f0027777cd
b21700064c0443d15ca68618eb49210a530ee77e
8658 F20110318_AABSEG woodling_k_Page_011thm.jpg
4fe371ac9db43511ed859a76fc9a82f5
ee3f95b12c5d3d63c9e668e16d1d8cd3f3402d5a
98405 F20110318_AABRZB woodling_k_Page_047.jpg
85a02856cf4dbc9f0f81159ba77c19b8
88d7af49da9a719beb186419bfbcccaaa9fd0ce0
1709 F20110318_AABSDT woodling_k_Page_004.txt
65ab2dc1acb60d54716242ddbc4ae288
82239fc8cbe8eaad680fd1370bbbd85d04b408bc
23482 F20110318_AABRYN woodling_k_Page_083.QC.jpg
1b0286deaf5e304fd466605940f1ba98
aeef7ea93f3558e9db8b73ee9a2edbe5cedc6604
43513 F20110318_AABRXY woodling_k_Page_056.pro
7b0b675c8d55a70941c1dc691505ee33
6a2ae2a730c82de232c8e6a10372f68f669964e1
101178 F20110318_AABSEH woodling_k_Page_066.jpg
41d191bcbacc2bd2caba008635a09904
246e430fc3395279b2df83066621852b2a3d5ce4
6382 F20110318_AABRZC woodling_k_Page_080thm.jpg
ee0797012886d26be142c1fe7637a26f
9fdfde05404e7630875d3fe1b30a5c5575e72268
6555 F20110318_AABSDU woodling_k_Page_145thm.jpg
29361f409bc31fdaa5938166f993b402
58ca8b079718e8e2d6161fc20fe1a31bcd3d2e3e
104854 F20110318_AABRYO woodling_k_Page_137.jp2
e015e3faae6fa7fa6f3beb0fd6ff38e1
d57ed84993c066ede7ea5c4fc31dfb2b3466adc4
26145 F20110318_AABRXZ woodling_k_Page_097.QC.jpg
0efa4c56a2c43f2898845b3eb81e53e1
a94e079e0c5869738c0a7a53552e24a083d1246c
51874 F20110318_AABSEI woodling_k_Page_108.pro
fa51f1332535593766e0657072e005db
4b463e84a54757f0ab9159041fa578852b003fa8
34029 F20110318_AABRZD woodling_k_Page_106.QC.jpg
8407882c0d10a85504a2020a31116719
9219dfd602969fa922981645f6649c1ff24f6294
101720 F20110318_AABSDV woodling_k_Page_032.jpg
13447bb03f80c99ba7e3a81a7238129a
8e0c9777a13542fa63285a9690cd6cd857ac4d5a
87833 F20110318_AABRYP woodling_k_Page_004.jpg
8dcb957dfb9a3628155da918015bd17f
2f3c5373ece2a3da1cc2086a0eca36d08ff55dc2
929162 F20110318_AABSEJ woodling_k_Page_075.jp2
ac0fc4916e877120b9373a20171076c8
1ecc6fccdb399d3ce831a43c5f85a3dec5976b99
45484 F20110318_AABRZE woodling_k_Page_139.pro
d7add63c5a7540fa3ff65a779de33484
ddb701336619fccf3a01b1359bae196d9a645464
F20110318_AABSDW woodling_k_Page_123.tif
28951fb273589c68a0b7cc3c77daf58b
6d430f7e9e2ac23a38ae7006fffda04b246e1c6a
41229 F20110318_AABRYQ woodling_k_Page_057.pro
ac1c0c3f0a9aeca1cb73215ca4dcf0cb
eed1cd79db5858bc529e44c339f16b7fa742104b
8167 F20110318_AABSEK woodling_k_Page_021thm.jpg
2cc47030457a3665b635f172c62f4ea9
33328a1ec579116bb56e9c18f86d0a85c41b0e42
6827 F20110318_AABRZF woodling_k_Page_096thm.jpg
190aa0c279ec728f66bda4a0091bbd41
03ea72bc7875e730d6792f32694b84c463686d6f
8005 F20110318_AABSDX woodling_k_Page_124thm.jpg
46674d8dedcb198a5de31977c0b9104e
8661c64451a4d2bcd2a34deb8d18f69a219dec33
8341 F20110318_AABRYR woodling_k_Page_027thm.jpg
e5f1e779d34649be3816617ced711b80
5f82427de0d1cf5eb1a72b20b4c94f58b9744da1
7010 F20110318_AABSFA woodling_k_Page_111thm.jpg
402d39474ec27c2b33196f18192675ec
cc1c1a4445d4b3f33c5c811c71d3caaae4a9fc53
88681 F20110318_AABSEL woodling_k_Page_084.jpg
05ff446f54a9ac8e89e42d11fe1921d6
b679fce1f5fc6fd3dbb39bcee708eba0954f45b4
F20110318_AABRZG woodling_k_Page_100.pro
3bc3e9525be96ce3312ce92fcdb78fbd
85791e14691af6cf8e7f84ad4df72189f08c2655
107510 F20110318_AABSDY woodling_k_Page_101.jpg
5d4d12427a8d68a754f28430cdce4a64
a68bf2c5f100519dc7d751d2a6051f8564cbcfcb
107857 F20110318_AABRYS woodling_k_Page_086.jpg
716039f661644159bd167804c706109e
451e80562356fdb93593497a625ac86c2d6f4c99
7633 F20110318_AABSFB woodling_k_Page_089thm.jpg
f6ade9e5dbd29000747daccf6c42feb2
0f28d9834c77589fea77890752b6c00547ec502d
858436 F20110318_AABSEM woodling_k_Page_013.jp2
b2558ca39004c9601d4d85f14122c8a9
65676b4d4e926664dd798cae8a52f601b3be8767
7261 F20110318_AABRZH woodling_k_Page_068thm.jpg
e908b17790cec763bd515ba0c7f37b94
3f204a4a85dd497bf4d568b2137f874a84b0b74c
8069 F20110318_AABSDZ woodling_k_Page_018thm.jpg
911a097fa80b627be88163b1f0700687
efb6d38726698b3c73b9afb5dd72d25d38a26cea
6059 F20110318_AABRYT woodling_k_Page_076thm.jpg
df0980f889f543eeec008cd994049cfe
2778d8ad6c687815ba09a8405ad9c5efe2d0f4b3
8223 F20110318_AABSFC woodling_k_Page_138thm.jpg
1f667ddec88da294d875c8c9129da3a0
7d6146c39c7060e4155548628a93a5f97ca3fd25
F20110318_AABSEN woodling_k_Page_069.tif
6044668962007a228ec5b0078b6a72fa
df992d2aea28ec900d632b73824ac976f3bc6204
29764 F20110318_AABRZI woodling_k_Page_067.pro
6e84f9e3328c3cce10485afc31aa4a3b
672748e8db661b8492a6f4c32a22720c77752277
F20110318_AABRYU woodling_k_Page_058.tif
22e9ed21d58aba1b15f6b6e6b7863e4c
250cea5271f619246661cbc51515165ddc11d78e
24269 F20110318_AABSEO woodling_k_Page_049.QC.jpg
2614eeb6557bc4c66d7531094a35bea4
da0d7231c024b0ad3e920b74afa892ffad0a9aea
107226 F20110318_AABRZJ woodling_k_Page_133.jp2
80a12d3f45d6c9e50142963395a6d102
236c3fe0752f2f38e6943abb2b53862e6aef9b6b
1985 F20110318_AABRYV woodling_k_Page_069.txt
b3033987e638986d6001ee47e500e811
35a91d5d08a5db0217963befccbd44a6ad0846c1
50319 F20110318_AABSFD woodling_k_Page_132.pro
2372101c533ec725ed693fc200f0cd75
6a44454ffd5a28b8cc1abeb1f9d87e8fe0b54b83
33314 F20110318_AABSEP woodling_k_Page_115.QC.jpg
d7edd11b76bf468dc63774fafb7a3a5b
eb5d12ad556506fd90b51c2e647954b9084486ac
109984 F20110318_AABRYW woodling_k_Page_020.jp2
084d1024d57822e46fe51d0450bb8e0d
679e8c0be79e2e2ce49a64e5c757f9e8fe2afcd8
31471 F20110318_AABSFE woodling_k_Page_076.pro
6fc8a8a4302350f807331c8bde0b73cb
ee08ccfd6747d765fe860a1ff91cbcdd160dfa74
6728 F20110318_AABSEQ woodling_k_Page_009.pro
5a33ceb7e09a6148a82fda1a3f5a1d6f
11143fe52d4c49e0393e30d8bdbe13f3349273c5
77216 F20110318_AABRZK woodling_k_Page_050.jpg
bf86466868eb871cb794d7ae333b837f
ba554a48d17287f6f1e8d3fafe62664a7286d60b
27244 F20110318_AABSFF woodling_k_Page_095.QC.jpg
2655cbf87beac037b196984873230f5f
792ec960a902a23457b9e6f9e08b059716227013
8227 F20110318_AABSER woodling_k_Page_072thm.jpg
452af86ce9f25f50258cc9531f5ab1af
37314eb4d556592c426415638ac4a65fa589f338
28308 F20110318_AABRZL woodling_k_Page_019.QC.jpg
e20bc6363b7c780117fa7a2b78fe1d69
a71b9c97c1084d254c725abea1128b1cd74c5186
86292 F20110318_AABRYX woodling_k_Page_022.jpg
de34c495cab4bd9bbcaa4f365757cb95
d8c4b20d6be37355471f4dee19db8bb0f8b76559
26312 F20110318_AABSFG woodling_k_Page_111.QC.jpg
afc833607f986224ad333183e49ee7a1
a9aa970c821b63a0fdad7d2a6adbcc26b7558830
1329 F20110318_AABSES woodling_k_Page_067.txt
83a681f222c6472aff2ccc2aae54a62b
1f42a450e16a89ac05b0ee8d39ecaf0928a2d414
33425 F20110318_AABRZM woodling_k_Page_144.QC.jpg
6e4fcf6fbeecd53a019516792a2566ef
4990167ac61655d3e9df0af8f6d68e10064ce982
F20110318_AABRYY woodling_k_Page_060.tif
dc22fd06c644a081fd77093b0dc52003
896b3ff8f0513ecb1fee3612b3203bac3f66b33d
F20110318_AABSFH woodling_k_Page_078.tif
d11906ca9ea1594575d0cb13ad65615f
9fbe2895e164523b7c1ffe9faa80c123537c9402
F20110318_AABSET woodling_k_Page_033.tif
8d97053b05a6755b7c999e3161157e75
ec78a54dc9ed44ec28d1a3efc719aa9088a4ccde
87964 F20110318_AABRZN woodling_k_Page_089.jpg
0d128d8b86bf0bbdacf80e0ec5fdabf1
3223bfb544ef78d65dc9350cfd1e90df42dac968
14006 F20110318_AABRYZ woodling_k_Page_013.QC.jpg
51f62b9c0bb1ddaa22b82dc267f1fc3f
40b8a1ef46deb64dda0b36d746337228c9e8d661
F20110318_AABSFI woodling_k_Page_040.tif
ba77652367dc1edb69f6fa1a27c8e44e
66df9dabb053e2ab5c5882e0198401ebf8beb5ac
50167 F20110318_AABSEU woodling_k_Page_021.pro
df02d6e3b274d867542a5fc27bd36611
61e53929a10b75717442b73657804259b02d1c5a
F20110318_AABRZO woodling_k_Page_007.tif
f4f0fde806ae609d0d86036edfca8832
135381a9fe1121b78d920647bce38b7578486220
6800 F20110318_AABSFJ woodling_k_Page_058thm.jpg
d74581f7e7543f19c457fc9da8b5f13f
8f269920f9ea7c6d7659b36394cd2774278e1502
26819 F20110318_AABSEV woodling_k_Page_048.QC.jpg
920a8f9fd468d15ea6751705f32e398a
04b2fc1378193557406fd73b8f14df34a7dcef6e
74193 F20110318_AABRZP woodling_k_Page_099.jpg
89589a7263c5a5c78c3f4209746dd033
484207307bce431433849fdd8bc28c64fa576fea
2565 F20110318_AABSFK woodling_k_Page_011.txt
7c6a735c510e91274dddddfe86e930e0
379f35ab7e281c227fbf66a10d29f674d66c9841
91696 F20110318_AABSEW woodling_k_Page_117.jpg
218023922961cdb76527f647b58c79e3
1a63cc0c93ec2c46b73c395fa8061cab5a2bc316
36945 F20110318_AABRZQ woodling_k_Page_050.pro
5240a5d207e19144e22a991afed05ee9
6409712aae6bcdb394c7b8b8b285c20722f3d5be
42541 F20110318_AABSGA woodling_k_Page_103.pro
bb1748560a4aac26bb8bb760a5ece7ed
e9dfe0d7f7e39e084b07c230f9adcf7599ab4d92
1782 F20110318_AABSFL woodling_k_Page_064.txt
294dced03f237f6adfbf563f92439e75
76b6aa58178ecef88d7720f0d1bc67a341c4c45a
6534 F20110318_AABSEX woodling_k_Page_079thm.jpg
5656a0f20e97601ab1527a226b527af3
394db4172d982aa955abe3c83f789ed1b3e96927
1906 F20110318_AABRZR woodling_k_Page_070.txt
93bdc559e764f894b8afd1e84f3b19d4
0c08d154784bda057c321c2ab33c200e096ec39c
1978 F20110318_AABSGB woodling_k_Page_017.txt
22918ed9f03873d5123b9eb4272d4fc6
071e993e6f1c5c4cd826e6145180f535813b31ab
51921 F20110318_AABSFM woodling_k_Page_023.pro
9a5a96958889409e303a7597eac4296b
c344179bfae061fd6790702247bc34bc5a44cd66
32046 F20110318_AABSEY woodling_k_Page_140.QC.jpg
828c680c6964ca51ce6cdc70fd6dd688
0bc5b41108e05124ce3b9653b685f56f08cd9596
106621 F20110318_AABRZS woodling_k_Page_119.jp2
c44e04149902423117d02f2ceef3e660
e8e105a1bf6149899bfc413c5f5eeefe88f1f7ec
49757 F20110318_AABSGC woodling_k_Page_060.pro
e273ebcbafc55ffd943bad9e4abb08d2
ac9f92069a8a8714f44a9d90b4d1629ef2a74c93
7677 F20110318_AABSFN woodling_k_Page_090thm.jpg
25f8891008a0b38db37556b88fe2f8ef
248f5cf6c5de532d66fe1bd78c1bd0f41a970c11
7508 F20110318_AABSEZ woodling_k_Page_059thm.jpg
f48cb2775bf479d301ac53564eed35f4
4e26b11fa7fbcc1f11c40c747bed5ed08bb57c27
112223 F20110318_AABRZT woodling_k_Page_037.jp2
5a4f38e75bbf193c8a8b16250afc4fca
c5b0c4282075066326872d6ccb2136e53b330871
104871 F20110318_AABSGD woodling_k_Page_030.jp2
8fae4687f2f6ba910b358b8ab3f189fc
3ea42640167de159a84f5c45f374ad3c0176b561
43674 F20110318_AABSFO woodling_k_Page_114.pro
db30931de5b96df797d270ba2ae8cfdf
f0b82e11925ccd26f45f9d6036e4bc087ec5e89d
1888 F20110318_AABRZU woodling_k_Page_026.txt
653dae941ff64d0cf4264b7688a6db0c
848e736596ff40009aa3df13cfcf7c7a4ec3eab6
52530 F20110318_AABSFP woodling_k_Page_024.pro
7d04ad67dbcee240083700130103a601
3f583b7e5379c930f7dd6d1e5d61772fa516ffaa
F20110318_AABRZV woodling_k_Page_011.tif
fd530731f3d24756a65517d6fc5690fc
1e09567f2f1a4d8373e7591062161e62b3826e1a
30053 F20110318_AABSGE woodling_k_Page_127.QC.jpg
8fa30c449e97ae61c007e1f605a52099
20eaa73f7a97d30acc3029f68146572716f8dfce
F20110318_AABSFQ woodling_k_Page_044.tif
f5151e576d8b9872bbb7989100eabbc3
fbabdffbd313fe34d620faa47aabcfe0dbaea9a6
2020 F20110318_AABRZW woodling_k_Page_036.txt
4f421940ecd5bb467979de82aa24c385
aa7a71c6180540a71d8bc0cf9f1a28cc8c407330
F20110318_AABSGF woodling_k_Page_137.tif
e4eebcf05afa94026a4e4bf4def92f3f
f1940351860d566d003cb908a9c8c8c3198e2b87
F20110318_AABSFR woodling_k_Page_085.tif
66d77deb966c7427a5671764507bf9bd
016944d799ecc88790670a35c4927bd42e5ccac4
F20110318_AABRZX woodling_k_Page_106.tif
0ec5d937a79aaba5fc7d20e985da7662
c816a291406c0eb6bc1a70516c83451a3f0e29e4
6778 F20110318_AABSGG woodling_k_Page_046thm.jpg
1baad28ef4fbc5bea5c4639dedecde4e
b16b12caac99f768bf58d2e8259f84fcc5e43bbd
7410 F20110318_AABSFS woodling_k_Page_010thm.jpg
bb93cc4f9718941fa6f9e0572b2e3d85
4114fa600539e3800ad20eeb8d843a33ebb9806e
F20110318_AABSFT woodling_k_Page_024.tif
c39dd68906948b254b7a1f786c2259fd
24386bf642e35a2f057f3174eebcbebaf58e6b8c
26899 F20110318_AABRZY woodling_k_Page_098.QC.jpg
b1f31e9dd23d1fedb54754f1aa1f7057
3ca92c2ad7a7f1c473fc3fbd9419efe76545d485
34724 F20110318_AABSGH woodling_k_Page_036.QC.jpg
47612b312b29a30ea69c20374dc015a8
020a7b3d6b008c173950087194ff2b38561d0b9c
23847 F20110318_AABSFU woodling_k_Page_015.QC.jpg
3bb9939fb27b775e67ab474e5282046d
0b8de108f29aac98d98c222428edaa1ae87380d9
7050 F20110318_AABRZZ woodling_k_Page_095thm.jpg
9d006b76c420b9de9d1fde1d65e1f418
b9a295ff6a2697a8769741dd2fc87d5a4a8ef745
47986 F20110318_AABSGI woodling_k_Page_107.pro
8f6f095b9556ff977f804901223ed1e0
6832b1b73d970bccc577b6d2338a9273e293bf04
8047 F20110318_AABSFV woodling_k_Page_085thm.jpg
5ac5b1e83f51aa2fee4a9465f6db9c4d
3701b2f4f1c5de27c7a27cc584b1a5676131feee
F20110318_AABSGJ woodling_k_Page_136.tif
0eb53bcf82ba8b0555fc268d9ebd36b9
169bc86d274518d95f8ad7c822feb4d298f377ea
6407 F20110318_AABSFW woodling_k_Page_034thm.jpg
9987f2a095e0b4964e79db52ca53095a
d191a19302b6427b4e5525e18dd90161701ee1ad
98265 F20110318_AABSGK woodling_k_Page_025.jpg
44086be09205e238c985a51a27ce7bc1
6a5b73ac35605d2d294d4ab537e2d76674bfb34f
53116 F20110318_AABSFX woodling_k_Page_144.pro
613b432711a9dc9fb7e26a8d4506a142
dd6d7eb799dcc55f136052a5d35ed003fcfb9ce1
2025 F20110318_AABSHA woodling_k_Page_062.txt
41d5ad5467d4f028f09b848837a40dd6
df4b5b0daab95483eb7c48d013da3c49f1822f3c
109719 F20110318_AABSGL woodling_k_Page_040.jpg
4bf4a039adcfb2c4676ff897b451b3c3
1929556531333cb7a99b46322490e95eb5f7b77c
107233 F20110318_AABSFY woodling_k_Page_102.jp2
b77608f438097dc2721a4e13390b6c7b
e8b7e9facfb1f2e18ccd5608d73e19469badcb9f
9326 F20110318_AABSHB woodling_k_Page_012thm.jpg
5fe9784ac0253980e0842fb6a105d65b
9c4c80e44aec1db5fbbbf46d98250f7f97ddb580
8148 F20110318_AABSGM woodling_k_Page_137thm.jpg
a262fc77636be4932aec26997ac9eab1
d69c084c0e06c784ed35b0ec407ce6917e2425a3
F20110318_AABSFZ woodling_k_Page_050.tif
35fea8993735f3e5640cbd88f38f0fd8
2f22ee4f4bd505c68b9196a3a60674a55effb1e0
F20110318_AABSHC woodling_k_Page_074.tif
0f41b831a51ce8848b41b9c757e2d652
0dfc4b418d0845abf6363dbd9cfdb24474abd7c1
1960 F20110318_AABSGN woodling_k_Page_134.txt
fe6233e3b986587ffe206d7a96198119
37fa119602d9fb05061ca957ac88a420fc043c5b
F20110318_AABSHD woodling_k_Page_105.txt
59709bdf22920bbee2a7905df3d30707
b6f4c00619f5773a23c7c6331422ec3fb79e2f23
1586 F20110318_AABSGO woodling_k_Page_110.txt
79db498db41a4524f2725b120fdecb12
c124bb37ac0004a8776e8d054ebb15c087819505
80743 F20110318_AABSHE woodling_k_Page_081.jpg
f7364bcec06b7013045bc680b668ffb8
e717d66403b4f1fb72264d564bfcddbaf4894b93
7725 F20110318_AABSGP woodling_k_Page_056thm.jpg
031cdf8095734f458d12c84c02350a45
9d90241b8284be2b3f0314bed25216970af5e337
34552 F20110318_AABSGQ woodling_k_Page_024.QC.jpg
f0aa5aa1dd8d1871e571e378c8719cb1
c4546e6f79b1e76be3622121e9cf5e35c6ee1c9e
7771 F20110318_AABSHF woodling_k_Page_125thm.jpg
827dc54265ce56bdd5b9de09633ce105
7ac0f95997c1c07b775566fdb15adbb3c0529bba
1236 F20110318_AABSGR woodling_k_Page_079.txt
11362b1fcac103bd47231a83fab3dc1e
cae55511311331608695dd605e663526a47afefa
32810 F20110318_AABSHG woodling_k_Page_017.QC.jpg
ba2457ca57257e8f0b864656d76424f8
2663de979baf9de4fdac407fe63464b2f85bd01c
1851 F20110318_AABSGS woodling_k_Page_043.txt
94a0abd5b31c486a23663c504bd39e3f
b848210f632fb1197f4c716f1bcd5b47bda70ab9
F20110318_AABSHH woodling_k_Page_107.jp2
36c38254b71e6580c7c776b32bd080ca
8b2cfc12212debf67cef816770ba41038b9d4391
F20110318_AABSGT woodling_k_Page_091.tif
c424ee9c4591e96368b086d006c8cfff
211e54c25651cff44986839da80547f6e2bc9981
35438 F20110318_AABSHI woodling_k_Page_007.QC.jpg
f7663fdd4219bea3a02efb0ff5fff383
1f4229b92d94625399fc5b8214966260139b9d57
F20110318_AABSGU woodling_k_Page_107.tif
cd52b1818cbf35769344fba7ae06b8df
b57719265e92685b1dd9f878e6a03edd0c12ab59
72630 F20110318_AABSHJ woodling_k_Page_076.jpg
cff281dae116a36839c82ade13d7e000
d40cc02ba2e9dc0135710bb1a3f06962230c9125
1051881 F20110318_AABSGV woodling_k_Page_054.jp2
0b90ec839335bb19308ca3839bc96758
91ed9869ff5bc4c4c2604ee910bb91128b65ab63
F20110318_AABSHK woodling_k_Page_132.tif
8f3b0394ef2a0ae2c5e5204444a96a7f
993407932d6170355f6555919823ca781e4b438c
7256 F20110318_AABSGW woodling_k_Page_053thm.jpg
01b6abec535e456363380c22c1a9a9ee
ee6fdb0bc56d32a393b8021d74737b36ac8cc72d
90003 F20110318_AABSIA woodling_k_Page_090.jpg
53c61befd614bd064cf22c5e6480fe75
3db0ca2afd174984670c5b22806e13750bac0a20
8422 F20110318_AABSHL woodling_k_Page_031thm.jpg
4e147e8d94cec17deabc381320b96383
2a20a787ff56abc62a589b5b4f79161fa7b48d7e
80997 F20110318_AABSGX woodling_k_Page_121.jp2
f4ffc7e3a8aa5dd1875493f906ab07f2
deb15c296aa71700b6e983b9d2ee6945c66f9161
2384 F20110318_AABSIB woodling_k_Page_010.txt
0a86477667050fe581f65f8451e09816
e53ec0e6f6a423d7b85470372d2139e462661c48
101390 F20110318_AABSHM woodling_k_Page_118.jpg
bdf20069d73c014251ceb4e67cf4e5cd
44da6a06b70abf2239fe4360940b321cce5a170e
F20110318_AABSGY woodling_k_Page_097.tif
752472a8b0d01a5fe3ea4f02b84bb0c7
e6e34a22548503c0fe08c34e5155d66356ffb198
32808 F20110318_AABSIC woodling_k_Page_097.pro
c1730bdfae8a0e778b5098492278e27a
23f87e632e3d02c332e7a4ade9bce3ae65137569
F20110318_AABSHN woodling_k_Page_019.tif
8e89d8d74ca677ec59ba4950453c858d
aff1a93dc46c1490a5bd2e2f61f207b02f6ac51a
31391 F20110318_AABSGZ woodling_k_Page_046.pro
3a62ff4935d364290807ea508346c4f3
0019cbfb66567e4f1696bdd5ebb65b130279be9f
1926 F20110318_AABSID woodling_k_Page_111.txt
46ec2c5035130861fb647757dd099f8b
da533d06aac1db000a79feded7684da14188d1ff
799214 F20110318_AABSHO woodling_k_Page_080.jp2
b295cc1e1663e18e85ec80e8a029748d
24c8ba92cd7d2677ecfb534468af14bc9a332386
30418 F20110318_AABSIE woodling_k_Page_099.pro
3a0cfd0684215a7008ce6ff7e5e10c97
d6b0fd7653eec08af110f3db851ff87b3951b056
39173 F20110318_AABSHP woodling_k_Page_116.pro
d265e4de5359777e1d7ced959d02082c
a3379f25f0597a54531c9d8f8f509c73e9b8e98e
5761 F20110318_AABSIF woodling_k_Page_120thm.jpg
802fd71a4778de5e02e200e22e64f7d3
2d608d1b43157f12cfd5d91ad1a1cfb2857f447f
6945 F20110318_AABSHQ woodling_k_Page_022thm.jpg
21a0aedd6374d53fbd88c59a4d389b24
0eec81d6bd946a22f76a36d6a49e768e78116afb
782530 F20110318_AABSHR woodling_k_Page_046.jp2
9a029402f0aeccea77642e2d2d11a161
8522a601e34160fd08cd3b03461fe9a9711f9236
29484 F20110318_AABSIG woodling_k_Page_070.QC.jpg
2c090a07bb7720e70b713c8066920578
355c19e821df6d75051cdc89e44924fa7cf4d6ba
114438 F20110318_AABSHS woodling_k_Page_044.jp2
e22780254e87fd301313a41a502f18b9
3824277bfef40027e8cc5a25c61d8dae3910e7e9
34467 F20110318_AABSIH woodling_k_Page_027.QC.jpg
e5f4e71d2034a87bcf1a156ea2e22b7e
c9e3fc5dc7d0d33f3437def0142f53be032e8495
108874 F20110318_AABSHT woodling_k_Page_006.jpg
27deaf3770b430e2ca13f97168ecce6f
f32e1a519f4ef30defd3ad506d8cf3f9927506d2
2327 F20110318_AABSII woodling_k_Page_106.txt
9381d336136ad160436ffe22273ba365
2ab4394d4a8c9a747181c114d4a93fa29ffc6ffb
35287 F20110318_AABSHU woodling_k_Page_089.pro
3a612d420934a1db64de1c1d806fb909
17a121ae5ad63f456a4749636042af8d7a5824e9
2040 F20110318_AABSIJ woodling_k_Page_108.txt
36b9c34925144d4c76cead2367429326
74c24894137dc60dd9f0cbb8d950cd7f67f29473
89889 F20110318_AABSHV woodling_k_Page_056.jpg
b33ec5f3eb8636cedbead9cd496518a0
4983025ba13369f6da3ff6a32c7585acb4670f82
96750 F20110318_AABSIK woodling_k_Page_043.jpg
ebadcc24b3f275ea98849b56ee488c6f
f01a928aa7809927ff2d85a66b40398f399f5d4b
27285 F20110318_AABSHW woodling_k_Page_075.QC.jpg
77e34a3e26773ec9c26ce89366ced261
3d36e6d9134035113a6ea57272fcb4059e636f87
82158 F20110318_AABSIL woodling_k_Page_096.jpg
4a3cb3fef672ed48d98a49f954e5501d
5f524acbb79bf4e2b17324eb6df958211532d643
F20110318_AABSHX woodling_k_Page_128.txt
2ec2a6482976fe7f376c15b37ae4289f
e1eff8ecf29b56891683b563633b3e3f75b627de
F20110318_AABSJA woodling_k_Page_028.tif
7acf804f90b76bd2f28879a15beeff02
365c51b854a15088e7a6fd7095cc70b02aaa0e2f
32725 F20110318_AABSIM woodling_k_Page_085.QC.jpg
52c981da5ec6e202cf0cd10335551ea1
774f3c9f0292e566294971438f43db493c664f85
23133 F20110318_AABSHY woodling_k_Page_079.QC.jpg
bb31e3ee09484c6c30e43d640d280e9d
66b63b5eac38e488198377917db849b353bb8128
1963 F20110318_AABSJB woodling_k_Page_102.txt
f7ffe2dcc4f71daca87e83f1c0ca93e9
2da4f5ae52e6557922149e0a14773eaf2aa54392
84867 F20110318_AABSIN woodling_k_Page_048.jp2
08313e20be21c07250e525a3c4f809e1
dd358f2cf10979a381f1c7793f0e7858c864805d
F20110318_AABSHZ woodling_k_Page_115.tif
b07bba78cf561cd3facb34f344f28f61
8b6e4e1ec506ad98a39a0c0e7affb1b5774fb484
4972 F20110318_AABSJC woodling_k_Page_002.jpg
da3deccfd131f7fcedaa039467d28365
479d973fffef59672e126468fbc45201b897225b
98884 F20110318_AABSIO woodling_k_Page_029.jpg
cbf6025037cbb0a517dae66fda7d0dd6
d2859caf08771b6b06f7be18a5558d92a003b58a
49719 F20110318_AABSJD woodling_k_Page_102.pro
ca32f57f3c639d5375ac40965ff4ab49
8e832e7f7221976a5f41a86361de112cf3a3d1cf
F20110318_AABSIP woodling_k_Page_135.txt
2a1cc7999cd10697dec4e1aa32510672
f3c48d48f5d324859cfd97bab60a6d4afa22c0b8
F20110318_AABSJE woodling_k_Page_099.tif
38d1b4c107325626ea14295edb7622d6
8ac17015aa18ff73bac9c281d9b57ab6af93a69d
36171 F20110318_AABSIQ woodling_k_Page_044.QC.jpg
acdc92857b64d8fd6a3bbe33bc71c62f
a786cd67c6b2431c1a4f0d71d6c287207cb6f582
F20110318_AABSJF woodling_k_Page_121.tif
938ec605a91f625860b687e3bcaaba3c
235b5c8f772b88c406f13e741b02c7292dae378a
41260 F20110318_AABSIR woodling_k_Page_016.pro
9ee91da31cd0f20057132dca952c6c64
1f36cb82fc64ebcfedcc6534cb7cb4042c0f4c80
29483 F20110318_AABSJG woodling_k_Page_065.QC.jpg
934b9b3b5cf35453d1cfa12280c90dfd
34ecfe95019efd928054cc021f3ea53711c7a2af
F20110318_AABSIS woodling_k_Page_088.tif
0d47638a0a31276913a135137f26fe53
7391c950de183dd2d7e76edb9d86c91e76ad0cc8
F20110318_AABSIT woodling_k_Page_048.tif
f7271cf738ba0abbd98d6b915e198aef
1570a2b6db7d411ab16b4e63271bff40f5687dfe
1814 F20110318_AABSJH woodling_k_Page_114.txt
69615d34e16fa50dc5ff0d1c83a18632
8b6c1092c8d2ac9f1192cb119e0a96edac50da95
39758 F20110318_AABSIU woodling_k_Page_075.pro
a42104bdb5bc4700e7d1e1ad194c0cad
a0419930fffbc2c385bf888537c43204b51cf6b1
48287 F20110318_AABSJI woodling_k_Page_146.jp2
6b9b583920c1fadb18f0ced948c4da26
1db977277f046b5d2d1707fa311215c3d7e6c1f8
4344 F20110318_AABSIV woodling_k_Page_007.txt
9acb3560a2d715a865808794064afa82
dc1a35189250adc1a3e062f9cc18830bd72e771e
F20110318_AABSJJ woodling_k_Page_055.tif
a0b631709d17f6aed1068c23c7f60e64
82b1df6ce659a800997058923d84ba38630753b9
826003 F20110318_AABSIW woodling_k_Page_052.jp2
4848b2b0e32601f0746dcff230e510ea
cdbfe3f4f257824ad7eb0a90279360d8feb9d6f8
964556 F20110318_AABSJK woodling_k_Page_125.jp2
04810511d4ac98f38ead2ffab3a8a5ad
255e15873770387325650e1d30b655bf6ef75f2f
75160 F20110318_AABSIX woodling_k_Page_041.jpg
a0b0499bede32291c526b4bb3138b102
a30ba1696211d35e63d73f5880ee0fa506f27def
33100 F20110318_AABSKA woodling_k_Page_045.QC.jpg
e99c796a2019f6929f4a585c712c3cb2
dc008a1c78741c9f201c4488392d4f23bc68d61e
F20110318_AABSJL woodling_k_Page_144.tif
813367b9e19ca9481257507db76b5a9c
efcb5cfbcc9c98d6cbfd2257506c5900414c46e8
7827 F20110318_AABSIY woodling_k_Page_043thm.jpg
28718191a447d5125ad6ce023e6516d8
74d151c1c8065a583ff32b1557ba12bd96f2cfdb
1280 F20110318_AABSKB woodling_k_Page_002.pro
42ed0416adf1a3e4312c3d37c0a82770
f7b1ee1bf6faeb05c17454e801ccdce740044f31
30500 F20110318_AABSJM woodling_k_Page_082.pro
83d3214874b1cfaed9c62a12caa9cdc4
f7a038ceacd85380a20ad0444dc4dabe28d841ca
F20110318_AABSIZ woodling_k_Page_088.jp2
6fd7cb39e255ef1167931cba1fb203aa
27561c5aeef45e717627a1fa9d04945384a2e855
7882 F20110318_AABSKC woodling_k_Page_026thm.jpg
e6d64ce83942977aead06ee32b52e24e
5f99fa25e758560f392c25813cb11b4523f1adc3
2056 F20110318_AABSJN woodling_k_Page_023.txt
681848cabfe9b5a9e77b8d0ef68fbbc2
40b489926fdf98dc0ac3aa3f4314cb2fe4f75db8
F20110318_AABSKD woodling_k_Page_100.tif
37933276e900b986a1e4639f65ddaa30
795f326bf777fb81adfeb4684d18146bd5be83f6
81463 F20110318_AABSJO woodling_k_Page_057.jpg
d21eb9df7a563859b16c2b7668b162e3
7d365ff078d02ae33e4fc9806278a22e341aaaf7
37165 F20110318_AABSKE woodling_k_Page_077.pro
4ae50dffc079c83ef070f1774abf71aa
64b44833c7d41f57e31954cf4e7f333b951ac3b8
43244 F20110318_AABSJP woodling_k_Page_065.pro
69f617d71bc4d29c8c918f9ea13fab9e
d7f5f9c434373c98b37466b3706330485b0eedf7
1852 F20110318_AABSKF woodling_k_Page_056.txt
50f69ff4c63b9287ccfc6a2a6436ff1f
6cca45c707b797b3207ce0065932d60ce6e15a67
1934 F20110318_AABSJQ woodling_k_Page_019.txt
d4b3e630e745f144ba72da55584cd834
270ee4077204762eb073dea75a745de179e5e076
1332 F20110318_AABSKG woodling_k_Page_015.txt
d9adc8f275c6025c866526906928d947
cc1e27f7dffe1cf00540f6d404e09a9160dd4592
84374 F20110318_AABSJR woodling_k_Page_111.jpg
464a6278d342f8e0300f0134cd801bfc
188ffb5729394cfce1c070f4490225397bef9cf3
27962 F20110318_AABSKH woodling_k_Page_001.jp2
005dbf178b658ac8493625d63eccce7b
e18f37ff55185cbe79fb634d75e4a8253f50339a
F20110318_AABSJS woodling_k_Page_038.tif
7d46a4b9b3d33472b05d1210a1d84635
4710e0ffa80045cda4f24fe03318542a911d6b41
39439 F20110318_AABSJT woodling_k_Page_111.pro
3b9e87474f49a33ce2ff901f536a8d32
245d00e097664f8f5f68f7b09e9357df5abcac18
23722 F20110318_AABSKI woodling_k_Page_123.QC.jpg
a42cce0cae38325896e34f95fa8e2714
d321513385c5eb807c5b4aa7943be5c008ee872b
F20110318_AABSJU woodling_k_Page_115.txt
60e169756272a03e448e4bd345b66fa1
5efccae15463644cc4ca55dc41c3964a4fd4fca2
32893 F20110318_AABSKJ woodling_k_Page_133.QC.jpg
b9f801d9cd89bf80eb278bccfa38dbb5
1fc10898b7dcfa3c23d698e50652204650b8acb2
8244 F20110318_AABSJV woodling_k_Page_118thm.jpg
1f954ae270f98fd0ac14b6b68f316b47
1d4f63a292cd69f7a14bcfe26dcbc862b6d5342a
922732 F20110318_AABSKK woodling_k_Page_022.jp2
8b8553389d686301a88cf6dbf9f7db9b
a3e058ad78932ab17f6c7c56978c2c621ede2075
F20110318_AABSJW woodling_k_Page_145.tif
8e5ab7b3550ec5c427a934175e7fd557
9772115bc23f86687cecdc71f2fda7f282abf502
28411 F20110318_AABSLA woodling_k_Page_033.QC.jpg
aa30837059a90ae5d5b1b1c292f50081
d3297dd7e5106b1d3ce413ba9f32e47f88c33ce9
90304 F20110318_AABSKL woodling_k_Page_116.jpg
83ad762ce72b45322747eb7422bbf6f1
2cd0cc754ede71ca86b9b4214d249c1311cbc256
31871 F20110318_AABSJX woodling_k_Page_018.QC.jpg
250e4bfc22267006fbe679aded9d426e
c72c59c5524b359bf6e8e301c68cfb5f62ea3820
F20110318_AABSLB woodling_k_Page_032.txt
a03bc3cc5bfe2aa65baca07283c608d2
cd0909baf6215a018f5ab86eaad5debee6b190ed
F20110318_AABSKM woodling_k_Page_050thm.jpg
3f614b8061b33867a1ee24e6e43b03d3
3f643d1e9d8b92b8203fe74b7784f8eb758f7b43
1566 F20110318_AABSJY woodling_k_Page_097.txt
5c298543d228e8820425ac6ee8d3fbe1
ceb0824a2d54f5603267e25ca2f4f70a1c5f72e4
6886 F20110318_AABSLC woodling_k_Page_110thm.jpg
995ed8d932be5d9b72f96c72e884d686
5e43829d27eb4fea32e6d061833456845baaf4ee
8568 F20110318_AABSKN woodling_k_Page_040thm.jpg
672ce1235b48285889bda8ecd0180ab0
830571976eef98b389e62f7adf4d3fd44d3bf86c
52955 F20110318_AABSJZ woodling_k_Page_027.pro
914995c6eff97b6dbca6d9d1951b98e1
0e62597bca2798f2fb4c6d3b550938a80f7bdf57
8620 F20110318_AABSLD woodling_k_Page_106thm.jpg
d4e0e9eb6193dbded4292954b1ac67a7
da8f08d1c71b7092339d9ee013eb9d177121fcad
47802 F20110318_AABSKO woodling_k_Page_026.pro
fa4ef212f2bca24c1a24ba134d6fe3a9
265326e7c043165eef0182e116cb3e9519a0af10
1922 F20110318_AABSLE woodling_k_Page_119.txt
84d768071a58f239987c86414f40cd84
27f77408f19a5576af318fc4110ffcef7fae0466
8722 F20110318_AABSKP woodling_k_Page_024thm.jpg
b7b624e10e49fef5adb3e27077be3afb
208f413ffadcdab67b24497e4088ed7ff3452d59
101349 F20110318_AABSKQ woodling_k_Page_141.jp2
0f29cb7b2cd7200c468b54ab9db71862
9a6b559e343bce055a11052bddf9df2d80e2ceef
F20110318_AABSLF woodling_k_Page_104.tif
c45f8d2b209eaf793509b2fbd43c4797
f7abb0bf5b74ba976c0254bff321cdd00d07b7c6
41368 F20110318_AABSKR woodling_k_Page_127.pro
df72f1eec96ab395355e65bdb1d9b7e5
f50d80008596f2417f0c78abb82938832b6077bb
2051 F20110318_AABSLG woodling_k_Page_103.txt
a300bd5b1178c8932fed4f1936f71c57
092d0cef04ffc6cee9bc7548c39c63b34b9cd9a1
2375 F20110318_AABSKS woodling_k_Page_045.txt
650cc88ebd6eee3301cf206231330c33
0f9b41e5f730c471b16db7c080c094cf0a6d4c4c
7687 F20110318_AABSLH woodling_k_Page_114thm.jpg
b8bdc7d257c5cd5275c54bd7bc30858d
45acd860237cca04b33131a8f51fbcc31b859a5c
F20110318_AABSKT woodling_k_Page_052.tif
343648e222aff9d3983c37971f4bc28e
68136a1ffc6c39d5d05f4e70db3a9ed73793ef04
80006 F20110318_AABSLI woodling_k_Page_006.pro
f0d357fceff9e8731940a53c6ae201d6
4613428954380923018a5a2dd9d8a2073f150157
4255 F20110318_AABSKU woodling_k_Page_005.pro
3208773f936840466d207dd1aa4c1f5b
2b271b71acdefed80f006a32dd402ab29b271e8d
2123 F20110318_AABSKV woodling_k_Page_104.txt
83b3d89c7afbda3defc214341df05239
b4248ad04e6ca96e7186bc81a50581df775aa9a4
1723 F20110318_AABSLJ woodling_k_Page_009thm.jpg
45d17a9cca03bcb97c08913184bbbc7c
1688aefacb754ab1c10fff0e3d5d789ccfbe7a75
F20110318_AABSKW woodling_k_Page_001.tif
62ac91c1114230e9e7b3993b87ee3bf5
a5d2fbc91041568089e487b900fe36c5f79b724c
101784 F20110318_AABSLK woodling_k_Page_102.jpg
8ca0761125fd59793a673f9e9a28a838
f2ddb0cc074456bfc74cc9ca5c06818d630771df
33146 F20110318_AABSKX woodling_k_Page_021.QC.jpg
f83e0554db991c4611d85b3ce59bd386
282189a6ed801e8d1d09facab9c08fedbda7ee4e
7277 F20110318_AABSMA woodling_k_Page_071thm.jpg
9318e7c2e7ba007f3fa1cb08371c3724
7da376172019c00d6eae405ff143dba8e5161c83
1218 F20110318_AABSLL woodling_k_Page_130.txt
21e7dc571679e3649af80da92a7269cc
dedaf1be5e45d07f0516bbf925c5689956082e36


Permanent Link: http://ufdc.ufl.edu/UFE0012801/00001

Material Information

Title: Using Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry to Study Modified Polypeptides
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0012801:00001

Permanent Link: http://ufdc.ufl.edu/UFE0012801/00001

Material Information

Title: Using Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry to Study Modified Polypeptides
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0012801:00001

Full Text












USING ELECTRON CAPTURE DISSOCIATION FOURIER TRANSFORM ION
CYCLOTRON RESONANCE MASS SPECTROMETRY TO STUDY MODIFIED
POLYPEPTIDES















By

KELLIE ANN WOODLING


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Kellie Ann Woodling



































To MaMa and PaPa Murphy















ACKNOWLEDGMENTS

I wish to thank my family for all of their love and support. I am especially

grateful to my parents for always supporting my decisions and being there whenever I

needed them. I also thank my sister Katie. Her love and friendship means the world to

me. I also wish to thank my grandparents, Martha and Burlond Murphy. Without their

continued love and support, I don't know where I would be today.

I would like to acknowledge Dr. John Eyler, my research advisor for the last 5

years. His guidance and scientific knowledge have proven invaluable. His caring

attitude and willingness to get to know his students is greatly appreciated. I would also

like to thank Dr. Yury Tysbin at the National High Magnetic Field Laboratory for his

help with the electron capture dissociation experiments that were vital to this dissertation.

His guidance with the use of mass spectrometry as a biochemical analysis tool is also

greatly appreciated. I wish to thank Dr. Arthur Edison and Dr. Michael Bubb, the

collaborators on the MARCKS protein work. Special thanks go to Iman Al-Naggar for

the synthesis and phosphorylation of the PSD segment that was vital to this work. I

would also like to thank Alfred Chung at the University of Florida Protein Core for the

peptide synthesis necessary for the electron capture dissociation experiments.

I would like to send very special thanks to my best college friend, Martha

Ostenrude. Her friendship over the last 9 years has been wonderful. The long

conversations and our love of "The Golden Girls" helped me to get through my graduate









career. Special thanks also go to Genay Jones for the long talks and his willingness to

watch every college basketball game imaginable the last 5 years.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S .................................................................... ......... .............. iv

LIST OF TABLES ..................... .......... ..................... ............ix

LIST OF FIGURES ............................... ... ...... ... ................. .x

1 IN TR OD U CTION ............................................... .. ......................... ..

Properties of Peptides and Proteins ................................. ...........................................2
Peptide and Protein Form ation ........................................ ......................... 2
Post-translational M odifications................................................... .................. 5
Biochemical Analysis of Peptides and Proteins ..........................................................8
M ass Spectrom etry ............................................ .. .. ............. ......... 12
Ionization M methods ........................................... .. ....... .............. ... 14
Bottom-up versus Top-down Proteomics.........................................................21
M A R CK S Proteins ............................................ .. .. ............. ......... 24
O v erv iew ...................................... .................................................. 2 6

2 FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS
SP E C T R E O M T E R Y ......................................................................... ...................27

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Instrumentation...29
Io n M o tio n ................................................................3 3
Cyclotron M otion .................................. ... .. ..... ............ 33
T rapping M otion ............ ............................................ ............ .......... .. 35
M agnetron M otion............ ............ .. ....................... ............. ............. 36
Experim ental Operation of FTICR-M S ........................................... ............... 37
M ass R solution ..................... ................. ... .. .. ......................... 4 1
Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem
M ass Spectrom etry .......................................................... ................. 44
Slow Heating Dissociation Techniques............... ..............................................44
Electron Capture D issociation................................... ............................. ....... 47
C o n clu sio n s..................................................... ................ 5 2

3 IMPLEMENTATION OF AN ELECTRON CAPTURE DISSOCIATION
S O U R C E ...................................... .................................................. 53

In tro d u ctio n ...................................... ................................................ 5 3









E xperim mental M ethods........................................................................ ............... 55
Sample Preparation ................ ...... .... ... ... ..... ... ............ ....55
Fourier Transform Ion Cyclotron Resonance Mass Spectrometry...................55
Sustained Off-Resonance Irradiation Collision Induced Dissociation
(SO R I-C ID ) .................. .......... .................................................. ..... 57
Electron Capture Dissociation (ECD) ...................................... ............... 57
D ata A n aly sis................................................ ................ 5 8
R results and D discussion .................................................... .............. .. ............. 58
Implementation of a Dispenser Cathode for ECD Experiments .......................60
Comparison of the Electron Ionization Filament and the Dispenser Cathode ....62
Comparison of ECD and SORI-CID ......... ............... ..................63
Electron Capture Dissociation of Modified Peptides .............................. ....65
C o n clu sio n s.................................................... .................. 6 8

4 ELECTRON CAPTURE DISSOCIATION STUDIES OF A SERIES OF
M ARCKS ANALOGUES .............. ............................................................69

Introdu action ...................... ............... ....................................................... 69
Experim mental M methods ................................................................ ............... 73
Sample Preparation ....................... ... .............. .......... ....73
Basic Fourier Transform Mass Spectrometry ................... ..................74
Electron Capture Dissociation Fourier Transform Mass Spectrometry .............75
D ata A n a ly sis ................................................................................................. 7 6
R results and D discussion .......................................................... ........ .......... 76
Fragmentation of the Non-phosphorylated Peptide............................................78
Fragmentation of Singly Phosphorylated Polypeptides .....................................80
Fragmentation of Doubly Phosphorylated Polypeptides................................. 82
Differentiation of Polypeptides with Identical Mass and Number of
M o d ificatio n s ................................................... ................ 84
Relative Abundances of the Fragment Ions......................................................93
Reproducibility of Electron Capture Dissociation Experiments .........................95
C o n clu sio n s..................................................... ................ 9 8

5 ELECTRON CAPTURE DISSOCIATION STUDIES OF THE
PHOSPHORYLATION SITE DOMAIN OF THE MARCKS PEPTIDE .................99

Intro du action ...................................... ................................................ 9 9
Experim mental M methods ............................................................................ .. 101
Sam ple Preparation ................................................ ...... ... ........ .... 101
PSD Phosphorylation................... .......... ............................. 102
ESI-FTICR-M S Basic Analysis .............. .... ..... .. ..... ............... .... 103
Electron Capture Dissociation Fourier Transform Mass Spectrometry ............103
D ata A n aly sis............................................................................................... 10 4
R results and D discussion ............................... .... ............................ .............. 104
Fragmentation of Non-Phosphorylated PSD ....................................................106
Fragmentation of the Phosphorylated PSD Segment ............ ................107
C o n clu sio n s................................................... .................. 1 14









6 CONCLUSIONS AND FUTURE DIRECTIONS ............................116

L IST O F R E F E R E N C E S ......... ................................................................................... 12 1

B IO G R A PH IC A L SK E T C H ......... .............................................................................131



















































viii
















LIST OF TABLES


Table page

4-1. Predicted monoisotopic masses for the seven MARCKS analogues. .....................77

5-1. Monoisotopic masses of the unmodified and modified PSD segments................... 105
















LIST OF FIGURES


Figure p

1-1. Zwitterionic structure for an amino acid residue ......... ........................................ .3

1-2. Formation of a peptide bond from two adjacent amino acid residues....................4

1-3. Adding a phosphate group to serine to form a phosphoserine molecule..................7

1-4. Formation of the multiply charged droplets occurs due to evaporation of the
solvent from the sam ple m ixture ..................................................... ... .......... 18

1-5. ESI m ass spectrum for ubiquitin ........................................ ......................... 19

1-6. Bottom-up versus top-down proteomics........................................ ............... 23

1-7. The sequence of the intact MARCKS protein......... ....... ...................... 25

2-1. FTICR-MS performance features that increase with an increase in the magnetic
fi eld stren g th ................................................... ................ 3 0

2-2. Examples of a cubic trapping cell (top) and an open cylindrical trapping cell ........31

2-3. The magnetic field bends the ions into a circular orbit ........................................35

2-4. Natural ion motion modes of an ion trapped in an ICR cell.................................37

2-5. An example of a generic FTICR-MS experimental sequence..............................38

2-6. The ion optics utilized for ion transport from the source region to the analyzer
cell of a typical FTICR instrument manufactured by Bruker Daltonics ..................39

2-7. U ses of ion cyclotron excitation ........................................ .......................... 40

2-8. Example of the Fourier transform of the time domain transient to obtain the mass
sp ectrum for sub stance P ............................................................... .....................4 1

2-9. Effect of collecting more data points on the resolution of the resulting spectra ......43

2-10. Evolution of the ion cyclotron radius and ion trajectory during SORI-CID ..........46

2-11. C leavage of the am ide bond .......................... .. ............................ .....................46









2-12. Cleavage of the am ine bond .............................................................................. 50

2-13. Formation of a and y products during the ECD ............. ..... .................51

2-14. The cleavage of the disulfide bond can occur during the electron capture
dissociation process............ .......................................................... ...... .... .... 52

3-1. Representation of the fragment ions formed during the ECD process................. 55

3-2. Instrumental schematic of the Finnigan NewStar T70 Fourier transform mass
spectrum eter. .............................................................................56

3-3. ECD mass spectrum obtained for ubiquitin using the El filament as the electron
so u rc e ............................................................................ 5 9

3-4. Schematic representation of the dispenser cathode utilized for the ECD
experim ents ........................................... ........................... 60

3-5. First ECD mass spectrum obtained using the indirectly heated dispenser cathode..61

3-6. ECD mass spectra for horse heart myoglobin .................................. ............... 62

3-7. Fragmentation summaries for the ECD of horse heart myoglobin..........................63

3-8. SORI-CID spectrum obtained for the dissociation of ubiquitin.............................64

3-9. Combined ECD (red) and SORI-CID (blue) fragmentation summary for horse
heart m yoglobin ........................................................... .. ........ .... 64

3-10. ECD mass spectrum obtained for KIGDFGMTRDIYETDpYYRKGGK ............65

3-11. ECD mass spectrum obtained for GnLAGPnLQSpTPLNGARR..............................66

3-12. ECD mass spectrum for SNKSQKLLRpSPRKPTRKISK ............................. 67

4-1. Predicted ion pairs formed during tandem mass spectrometry experiments............71

4-2. The sequences of MARCKS analogues 1 through 7.............................................73

4-3 Schematic of the Bruker 47e FTICR mass spectrometer ...................................74

4-4. Home-built 9.4T FTICR mass spectrometer at the NHMFL................................75

4-5. FTICR mass spectrum obtained for the non-phosphorylated MARCKS analogue..77

4-6. FTICR mass spectra obtained for the doubly phosphorylated MARCKS
an alogu es ............. .. ...... ......... ........................................ 7 8









4-7. FTICR mass spectra obtained for the mass analysis of the singly phosphorylated
M A R C K S analogues ..................................................................... .....................78

4-8. ECD mass spectrum obtained for the dissociation of MARCKS analogue 1. .........79

4-9. ECD mass spectrum generated for MARCKS analogue 5 .......................................80

4-10. ECD mass spectrum generated for the fragmentation of MARCKS analogue 6 ...81

4-11. ECD mass spectrum obtained for the dissociation of MARCKS analogue 7 ........82

4-12. ECD mass spectrum obtained for the dissociation of MARCKS analogue 2 ........83

4-13. ECD mass spectrum obtained for the dissociation of MARCKS analogue 3 ........84

4-14. ECD mass spectrum obtained for the dissociation of MARCKS analogue 4. .......85

4-15. Fragmentation summaries generated using experimental peak list 2...................86

4-16. Fragmentation summaries generated using experimental peak list 3 .....................88

4-17. Fragmentation summaries generated using experimental peak list 4 .....................89

4-18. Fragmentation summaries generated using experimental peak list 5 ...................90

4-19. Fragmentation summaries generated using experimental peak list 6...................91

4-20. Fragmentation summaries generated using experimental peak list 7...................92

4-21. Relative abundance (RA) plots for the c and z-type fragment ions for
M A R C K S analogues 2-4 ............................................................... .....................94

4-22. Relative abundance (RA) plot for the a-type ions of MARCKS analogues 2-4 ....95

4-23. ECD mass spectra generated for a doubly phosphorylated MARCKS analogue...96

4-24. Relative abundance plot for the a-type ions of MARCKS analogue 2 obtained
in M ay 2005 (blue) and August 2005 (purple).............................. ............... 97

5-1. Sequence of the PSD region of the MARCKS protein...............................101

5-2. Mass spectrum obtained for the non-phosphorylated PSD segment ......................105

5-3. Mass spectra generated for the synthetic PSD segment .............. .....................106

5-4. ECD mass spectrum obtained for the non-phosphorylated PSD segment..............107

5-5. ECD mass spectrum for the singly phosphorylated PSD region of the MARCKS
p ep tid e ......................................................................... 10 8









5-6. Fragmentation summary obtained for serine 1 phosphorylation..........................109

5-7. Fragmentation summary obtained for serine 2 phosphorylation..........................110

5-8. Fragmentation summary obtained for serine 3 phosphorylation..........................110

5-9. Fragmentation summary obtained for serine 4 phosphorylation ...........................111

5-10. Fragmentation summary obtained for serine 5 phosphorylation........................112

5-11. Fragmentation summary obtained for the unmodified PSD peptide using the
peak list obtained experimentally for the singly phosphorylated peptide............113

5-12. ECD mass spectrum for the doubly phosphorylated PSD peptide. .......................114















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

USING ELECTRON CAPTURE DISSOCIATION FOURIER TRANSFORM ION
CYCLOTRON RESONANCE MASS SPECTROMETRY TO STUDY MODIFIED
POLYPEPTIDES

By

Kellie Ann Woodling

December 2005

Chair: John R. Eyler
Major Department: Chemistry

The phosphorylation of serine, threonine and tyrosine residues plays a vital role in

many aspects of protein expression and function. Protein phosphorylation is a reversible

process that acts as a molecular switch, controlling pathways in metabolism, signal

transduction and cell division. Because the phosphorylation of many proteins has been

shown to alter the structure and function of the protein, localization of the site of

modification has increasingly been studied in the last few years.

The use of many standard analytical and biochemical techniques, such as gel

electrophoresis and Edman degradation, allow us to identify unmodified peptides and

proteins. However, the use of these techniques to determine the sites of post-translational

modification has proven to be more challenging. Because of this, mass spectrometry has

been used extensively in the identification and subsequent localization of the sites of

phosphorylation for many peptides and proteins. The use of electron capture dissociation

(ECD) Fourier transform mass spectrometry (FTMS) has proven to be a valuable tool in









the localization of many classes of modifications. Unlike other mass spectrometric

dissociation techniques, ECD uses low energy electrons for the fragmentation process. In

this process, the fragmentation occurs prior to energy randomization, allowing the

modifications to remain intact.

In our study, an electron capture dissociation source was installed on an existing

FTMS system. Both an electron filament and a dispenser cathode were used for the

electron capture dissociation experiments. Once the operating parameters were

optimized, several modified and unmodified peptides and small proteins were

fragmented. The fragmentation efficiencies of both ECD sources were also compared.

In addition, a series of phosphorylated test peptides (MARCKS-related peptides)

was studied using ECD-FTMS. Resulting fragmentation patterns were used to determine

a series of fragmentation rules for the dissociation of both singly and doubly

phosphorylated peptides. The relative abundance of each phosphorylated and

non-phosphorylated serine residue was also explored. The established rules were then

used in an attempt to determine the site of phosphorylation for a synthetic peptide with a

sequence identical to the phosphorylation site domain of the MARCKS protein.














CHAPTER 1
INTRODUCTION

Proteins and peptides play vital roles in the biological world. Interest in studying

these biopolymers has grown considerably in the past 15 years because of the sequencing

of the human genome. The Human Genome Project, completed in 2003, provided the

complete genome sequence for human DNA. The goals of that project were threefold: to

identify all genes in the human DNA, to determine the sequence of all the base pairs that

comprise the DNA and to store this information in databases for use in future research

endeavors.-3 Once the complete genome sequence was obtained, the complete sequence

of amino acids for every protein encoded by the human DNA became readily available

for study.

Because of the desire to study the genomes of various organisms in a laboratory

setting, the complete DNA sequences of four model organisms were initially mapped.

These organisms include E.coli, S. cervisiae, C. elegans and D. melanogaster.2 The

model organisms are currently being used in laboratory research to gain a better

understanding of the protein content of these respective genomes. Genomes of the model

organisms can be much smaller than those of human DNA, speeding up method

development and analysis. For example, the human genome contains 3.2 billion base

pairs that encode over 30,000 genes, while the genome for E. coli contains approximately

4.6 million base pairs that only encode 3,000 genes.25 Each completely sequenced

genome then provides (ideally) the sequence of every protein encoded by that particular









genome. This has brought the study of the proteome (a science termed proteomics) to the

forefront of biochemical research.

Because proteins typically have functions that are important to the everyday life of

the cell, proteomic studies are rapidly evolving. Proteins can catalyze reactions, provide

structural rigidity to the cell, act as a molecular switch, control transport through

membranes, control gene function and participate in signal transduction pathways.6-10 In

the post-genome era, several challenges still remain. Although all the proteins encoded

by the genome are available in the myriad of protein databases, many areas of research

are still needed to determine the function and interactions of the proteins. In order to gain

complete understanding of protein functions and any post-translational events, the salient

properties associated with the proteins must be studied. These properties can include

protein abundance, the post-translational modification event, and involvement in

protein-protein interactions.

Traditionally, a three-pronged scientific approach is used to study the salient

properties. Separation science is used to separate and purify the peptides and proteins,

while analytical science is used to identify the protein and to quantitate the relative

abundance of the protein under certain conditions. Bioinformatics is also currently being

used in an attempt to manage the wealth of data generated. These three sciences are

proving to be rather robust in the study of proteins.4-9 The next section explains the

physical and chemical properties associated with peptides and proteins.

Properties of Peptides and Proteins

Peptide and Protein Formation

Peptides and proteins are composed of monomeric units called amino acids.

Although there are only 20 standard amino acids, thousands of combinations are possible.









This leads to an overwhelming number of peptides and proteins that can be studied by

numerous analytical methods. Although the study of intact proteins is advantageous,

many current studies use only a few representative peptide sequences associated with the

protein of interest. By studying single peptides associated with intact proteins rather than

the whole protein, insight can be gained into the overall function of the protein.5 The

peptide sequences are typically much shorter than those of the intact protein, making

analyses less difficult and time consuming. The amino acid building blocks each have

the same general structure (Figure 1-1). However, the side chain groups vary for each

amino acid. Linking two or more amino acids together results in formation of a peptide,

followed by formation of a protein.11,12

0
H3N
R

Figure 1-1. Zwitterionic structure for an amino acid residue. The R group varies with
amino acid, giving each amino acid different properties.

Although all amino acids contain the same general structure, the side chains (or R

groups) can vary in shape, charge, reactivity and hydrophobicity. Different properties of

the 20 different R groups cause differences in the physical properties exhibited by the

respective amino acids. Protein properties most affected by the different R groups

include polarity, acidity, basicity, flexibility, hydrogen bonding ability and overall

chemical reactivity. These R groups can be used to classify the amino acids into three

major categories: hydrophobic amino acids, hydrophilic amino acids and special amino

acids.12 The hydrophobic amino acids contain aliphatic side chains that make these acids

insoluble in water. Hydrophobic amino acids include alanine, valine, isoleucine, leucine,

methionine, phenylalanine, tyrosine and tryptophan. Normally, hydrophilic amino acids









are water soluble due to the presence of polar side chains. Examples of the hydrophilic

amino acids include lysine, arginine, histidine, serine, threonine, asparagine, glutamine,

aspartic acid and glutamic acid. The last category of amino acids is the special amino

acids. These acids each contain unique R groups not seen with the other amino acids.

For example, the side chain for glycine is only a single hydrogen atom. Proline is the

only amino acid with a cyclic R group associated with the structure. The side chain of

cysteine is considered unique because it contains a thiol group capable of forming

disulfide bridges in the peptide or protein molecule.

To form a peptide, the amino acids must be connected by a peptide bond. The

peptide bond is formed when two adjacent amino acids polymerize. The polymerization

reaction involves the interaction between the amino group of one amino acid and the

carboxyl group of the adjacent amino acid. Water loss is a consequence of this

condensation reaction (Figure 1-2).


H3N 0 H3N 0 H2 0
R O OH R 0 OH
R R

1
HH3N N H3N H
N
S0 N ..
O R -H20 R H R OH
R

Figure 1-2. Formation of a peptide bond from two adjacent amino acid residues. Water
loss is a consequence of the condensation reaction.

The resulting biopolymers can be composed of as few as 2 amino acids. Peptides

comprising only 2 amino acids are termed dipeptides. Oligopeptides typically contain

between 3 and 10 amino acids while polypeptides comprised of more than 10 amino









acids. Proteins are molecules generally considered to contain more than one polypeptide

chain. Proteins range in size from 40 amino acids to more than 4000 amino acids. A

three-dimensional structure is also characteristic of many proteins.11'12

Traditionally, proteins contain four different levels of structural organization. The

primary structure of a protein is merely the amino acid sequence of the polypeptide

chains making up the protein. The secondary structure of the protein is the spatial

arrangement of the polypeptide backbone. The configuration of the side chains is not

considered when discussing secondary structure. Tertiary structure refers to the

three-dimensional structure of the entire polypeptide. The quaternary structure is the

final level of organization. This structure is only used when describing a protein that

contains two or more polypeptide chains (called subunits), that associate through

non-covalent interactions. The quaternary structure then refers to the spatial arrangement

of the subunits in the protein.12 Although understanding the four organizational levels of

a protein is vital to the complete understanding of protein function, recent studies have

focused on identifying and localizing sites of post-translational modification in the

protein strucutre.13-17

Post-translational Modifications

Protein synthesis (how cells build proteins) is achieved in a three-part process. The

first step is transcription, which is followed by translation and events after translation.

By definition, translation is the synthesis of a polypeptide that contains an amino acid

sequence specified by the nucleotide sequence encoded by messenger RNA. Formation

of the peptide bond alone does not make a polypeptide or protein functional. Typically, a

post-translational modification event takes place after translation has been finalized,

allowing the protein to become functional. A post-translational modification (PTM) is a









chemical modification of a peptide or protein after translation. These modifications are

believed to play vital roles in protein interactions in cell recognition, signal transduction,

and protein localization. Current mass spectrometric analyses of post-translational

modifications include studies of the phosphorylation, acetylation, methylation and

glycosylation of the polypeptide sequence. 11,1316,18 While each of these modifications

may play crucial roles in the overall function of many polypeptides and proteins, only

phosphorylation of amino acids will be explored in greater detail here.

Protein phosphorylation is a reversible process that often acts as a molecular

switch, controlling pathways in metabolism, signal transduction and cell division. The

phosphorylation of proteins has also been shown to alter both the structure and function

of the protein. Because of this, the study of phosphoproteins has grown considerably in

the past few years.15'19-22 Currently, four different phosphoamino acids are known: the

O-phosphates, the N-phosphates, the S-phosphates and the acylphosphates. The addition

of a phosphate group to form any of these phosphoamino acids adds 80 Da to the

molecular mass of each acid by the net addition of HP03 to each residue. Formation of

the O-phosphates is the most common. The O-phosphates are formed when the hydroxyl

side chains of serine, threonine or tyrosine interact with the phosphate group to form a

phosphate ester bond. Serine phosphorylation is the most common of the O-phosphates

while phosphorylation of tyrosine is the least common. The phosphoamino acid content

ratio (pSer:pThr:pTyr) in eukaryotic cells is typically 1800:200:1. The O-phosphates are

often involved in signal transduction pathways and other cell regulatory events. These

phosphoamino acids are stable under acidic conditions, making analysis routine.13,14,23,24









Figure 1-3 shows a typical phosphorylation/dephosphorylation reaction for the formation

of the O-phosphates.


0 o
11 M ^u ATP, enzyme
H2N-CH-C-OH T H2N--CH-C-OH

CH2 H20 CH2

OH 0
Serine
Exact Mass: 105.0426 HO -P-OH

0
Phosphoserine
Exact Mass: 185.0089

Figure 1-3. Adding a phosphate group to serine to form a phosphoserine molecule results
in a molecular mass (monoisotopic) increase of 79.9663. A net gain or loss of
HP03 during the phosphorylation/dephosphorylation reaction is seen for the
entire class of O-phosphates.

The N-phosphates involve the phosphorylation of the amino groups of arginine,

lysine and histidine. The N-phosphates, often involved in a two-component signaling

mechanism in bacteria, result from the formation of a phosphoramidate bond between the

phosphate group and the nitrogen atoms of the amino acid. Although the abundance of

the N-phosphates is secondary only to the abundance of phosphoserine, studies involving

these amino acids are not as common as those involving the O-phosphates because of the

acid-labile nature of the phosphoramidate bond.23'25 The interaction of a phosphate group

with cysteine forms an S-phosphate. These phosphoamino acids are known as

S-phosphothioesters. Phosphocysteine is stable under most conditions but is rarely

studied.23'26 Lastly, the acylphosphates are produced by the interaction of aspartic acid or

glutamic acid with the phosphate group to form phosphate anhydrides. The site of

phosphorylation of these acidic amino acids is extremely labile under most conditions,









resulting in hydrolysis of the phosphate group during the analysis steps.23'27 Analytical

methods for both modified and unmodified peptides and proteins are discussed next.

Biochemical Analysis of Peptides and Proteins

Identifying proteins is straightforward from an analytical and biochemical

perspective. However, detecting and identifying post-translationally modified proteins

have proven more challenging. To analyze phosphoproteins, the low abundance of the

phosphoamino acids in the sequence must be overcome. The low abundance of

phosphorylation makes localization of the site of modification difficult and time

consuming with standard analytical techniques. Additionally, the phosphate group is

labile, further hindering identification and localization. If the analysis conditions are too

harsh, the modification can be destroyed or removed from the protein. Localization of

the modification becomes difficult, if not impossible, with the standard analytical

techniques.6'13'15'16'23 Many previous biochemical studies used a four-part approach to

analyze biomolecules: The procedure utilized must be simple and fast, the method must

be sensitive enough to generate the desired structural information, the protein of interest

should be readily searchable in the available databases, and the protein information

should be linked to the DNA sequence that encodes the protein. If all four of these tenets

are met, a better understanding of the overall protein function within the genome of a

particular species can then be determined.28 Previous studies have used Edman

sequencing, nuclear magnetic resonance (NMR), radiolabeling, and polyacrylamide gel

electrophoresis (PAGE) to study the modified peptides and proteins. Although each of

these biochemical techniques may give the desired information regarding the proteins of

interest, mass spectrometry has evolved into the method of choice for the analysis of

peptides and proteins.28-34









Edman degradation, or Edman sequencing, is used to sequence the amino acids

present in a particular peptide. In Edman degradation, the N-terminal amino acid is

typically labeled and cleaved from the peptide. The remaining amino acids, along with

the peptide bonds, are not destroyed during this process. The labeling and subsequent

cleaving of the amino acid can be repeated multiple times in order to identify each of the

amino acids in the sequence. The sensitivity of this technique has been reported to be in

the range of 5-50 picomoles. There are a few disadvantages associated with the Edman

sequencing procedure. The size of the peptide that can successfully be sequenced using

this method is limited to less than 60 amino acids. Intact proteins must first be digested

into smaller peptides before undergoing the Edman sequencing procedure. The process is

also a stepwise procedure. The entire process can be rather time consuming, depending

upon the size of the peptide being sequenced. In addition, the sequencing may not be

completed with 100% efficiency. The N-terminal amino acid may not be cleaved during

each subsequent step, resulting in incorrect or incomplete sequencing. If the peptide is

modified, the modification may be destroyed or removed during the analysis. This makes

Edman degradation less favorable for the study of modified peptides and proteins.28

NMR studies of proteins have been used to determine the three-dimensional

structure without the need for crystallization. Many current NMR studies are devoted to

the study of protein-protein interactions. X-ray crystallography is not amenable to these

types of studies due to the fact that many protein complexes will not crystallize. NMR

studies can give detailed information about the protein conformation (folded vs.

unfolded), the protein secondary structure, and the ability of the protein to bind various

ligands. The main drawback to the use of NMR is the large sample requirement. Many









proteins are only available in a limited quantity which may be very dilute. These issues

make protein NMR quite challenging.30-33

First observed in 1956, the use of radiolabeled phosphate to measure the

phosphorylation of proteins has become routine. The radiolabel studies can be performed

either in vivo or in vitro. In vivo protein labeling utilizes intact organisms, cells or tissues

that contain either [32P]phosphate or [y-32P] incorporated into the cells. The next step

involves the purification of the phosphorylated proteins without further modification by

proteases, kinases or phosphatases. The in vitro radiolabel studies involve the incubation

of a cell-free system with a protein kinase. The [y-32P] serves as the phosphate donor. In

both instances, SDS-PAGE analysis is utilized to separate the proteins. The radiolabeled

proteins are then visualized by autoradiography. These radiolabeling methods are very

sensitive and offer good resolution. However, in order to obtain specific labeling, large

amounts of the radiolabel are required. In addition, the observed

phosphorylation/dephosphorylation rates vary depending upon the specific residue that is

labeled, as well as the labeling time. If the rate of phosphorylation/dephosphorylation is

slow, the event may not be observed during the course of the experiment.34

Gel electrophoresis has been utilized in the majority of protein studies to date.

There are two types of gel electrophoresis that must be considered: 1-D gel

electrophoresis and 2-D gel electrophoresis. In 1-D gel electrophoresis, the proteins are

loaded onto a polyacrylamide gel matrix and subjected to an electric field. The proteins

will migrate down the gel based on molecular mass. The proteins with the lower mass

will migrate faster. One-dimensional gel electrophoresis does suffer from limited









resolution, making analysis of complex mixtures difficult. This challenge can be

overcome by the use of two-dimensional electrophoresis.29

Two-dimensional gel electrophoresis involves a two-stage separation process. In

the first dimension, the proteins are separated by charge using an isoelectric focusing

step. The proteins are separated using a pH gradient. Each protein is focused into a tight

band at its isoelectric point, the pH at which the protein carries no net charge. The second

dimension involves the use of SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel

electrophoresis) to separate the isoelectrically focused proteins by molecular mass. The

sensitivity of the SDS-PAGE technique ranges from 0.1-100 picomoles. However, the

analysis of proteins of less than 5000 Daltons (Da) is difficult. The analysis of low

abundance proteins also presents challenges. The concentrations of the low abundant

proteins often lie below the reported detection limits of the two-dimensional

electrophoretic method. Because of this, the low abundance proteins, such as

phosphoproteins, must be enriched prior to separation.29'35'36

Enrichment of phosphoproteins and phosphopeptides can be achieved with IMAC

(immobilized metal affinity columns). The IMAC procedure uses gallium or iron-

chelated affinity columns to bind the negatively charged phosphate groups. The

phosphopeptide is then retained by the column, followed by an elution step. These steps

produce a more concentrated sample that can be used in further analyses.37 Once the

proteins have been enriched, the standard electrophoretic techniques can be used. The

main drawback to the electrophoretic techniques is the time required for analysis. Each

gel run may take several hours, making high-throughput analysis next to impossible.29









Mass Spectrometry

Interest in the use of mass spectrometry as a primary analysis tool for peptides and

proteins has grown considerably in the past 15 years.7'9'10'38-49 The low sample volume

requirement and fast analysis time make mass spectrometry appealing for these studies.

Mass spectrometry involves the separation and detection of ionized molecules by

molecular mass. The sample molecules are typically ionized in the source region. Upon

ionization, the molecules are subjected to acceleration energy. Once accelerated, the

molecules travel from the source region to the mass analyzer region. The molecules are

then detected. The readout generated is reported as an m/z ratio, where m is the mass of

the ionized species and z is the charge on the molecule.50 Although most mass

spectrometers generate similar types of data, there are typically three different ion

separation and analysis methods utilized in the study of peptides and proteins. Mass

separation can based upon the time-of-flight (TOF) of the molecules from the source

region to analyzer region, separation by quadrupole mass filters (quadrupole MS) or

separation of ions in a three-dimensional trapping field (ion traps).9'39'5

In TOF mass spectrometry, the ionized molecules traverse a flight tube from the

source region to the mass analyzer. All ions are accelerated to the same kinetic energy.

However, the smaller ions will have a higher velocity, reaching the detector first. The

mass of each molecule is then determined by the time taken by the molecules to reach the

detector. Matrix-assisted laser desorption/ionization (MALDI) sources are often used in

conjunction with TOF mass spectrometers. The TOF mass spectrometer is well suited for

the pulsed nature of the laser desorption event during the MALDI process. In these

instances, a reflectron is often utilized. The reflectron effectively doubles the length of

the flight path taken by each ionized molecule. Increasing the time-of-flight for each









molecule results in higher resolution over time-of-flight experiments that do not use the

reflectron technology. TOF mass spectrometry is relatively inexpensive, compared to

most other mass spectrometric techniques. The resolution offered by this type of analysis

is often adequate for most routine peptide and protein analyses. Standard time-of-flight

measurements are well suited to most biomolecules. However, TOF mass spectrometry

with the reflectron technology is limited to sample sizes of less than 10,000 Da. The use

of the reflectron is thus often limited to the study of peptides rather than intact proteins.50

Quadrupole mass spectrometry utilizes a quadrupole as a mass filter. When an

electric field is applied to the quadrupole rods, only certain masses are allowed to pass

through the rods. The ions that do pass through the mass filter travel to the analyzer

region. The remaining ions (those which do not pass through the filter) do not reach the

detector. Quadrupole mass analyzers are well suited to high pressures, are capable of

analyzing samples up to m/z 4000 and are low cost instruments. Many peptide analyses

are performed with triple quadrupole mass spectrometers. The triple quadrupole

instrument contains three sections. The first quadrupole is used to select the ion of

interest for fragmentation. The second quadrupole serves as the collision cell for the

fragmentation event while the third quadrupole transmits the fragment ions to the

analyzer region. One drawback to the use of quadrupole or triple quadrupole mass

spectrometry is the limited mass range associated with the techniques. Protein samples

may lie outside the reported mass range of these instruments. However, the use of triple

quadrupole mass spectrometry for the study of peptide or protein phosphorylation is well

studied due to the introduction of electrospray ionization to the field of mass

spectrometry.50









Ion trap mass spectrometers have also been used extensively in the area of

biomolecule research. Ionized molecules are trapped by application of an electric field to

the electrodes of the analyzer region. Application of the electric field to the electrodes

allows the ions to either be trapped or ejected from the mass analyzer. This is

advantageous for tandem mass spectrometry experiments. Application of the appropriate

field allows certain ions to be ejected from the analyzer prior to introduction of a

collision gas. The resulting fragment ions can then be used to determine the sequence of

peptides and if any sort of modification event may have taken place. By coupling

electrospray ionization to the ion trap mass spectrometer, the possibility exists to study

many proteins and peptides due to the ability of the ion trap to produce spectra with both

good accuracy and resolution.50 A special type of ion trap mass spectrometer, the Fourier

transform ion cyclotron resonance mass spectrometer, will be discussed in more detail in

Chapter 2.

Ionization Methods

The emergence of mass spectrometry as a method of choice for the study of many

biomolecules can be directly attributed to the development of the soft ionization

methods.39'51 Both electrospray ionization (ESI) and matrix-assisted laser

desorption/ionization (MALDI) allow the production of ions from protein mixtures that

can be further analyzed by mass spectrometry. These techniques are classified as "soft"

ionization methods because the ions are produced with little or no fragmentation during

the ionization process.50'51

Before the 1980s, however, only "hard" ionization methods were available for

routine analysis. In electron ionization (EI), volatile compounds are introduced into the

mass spectrometer. A beam of electrons excites the sample, causing ionization and









possibly fragmentation. Although El is simple to use, this ionization process is typically

utilized for small molecule studies. The main drawbacks to the use of electron ionization

in biological mass spectrometry include the nonvolatile nature of biomolecules, the

decomposition resulting from the ionization process and the resulting fragmentation of

the sample prior to detection.50 In chemical ionization (CI), gas-phase ion-molecule

reactions occur between the sample and the reagent gas. Like EI, the chemical ionization

technique is most often utilized for small molecule studies. Biomolecule studies using CI

are limited due to the inherent mass range of the CI process and the need to derivatize the

biomolecules prior to ionization.50

Developed in 1981 by Barber and co-workers, fast-atom bombardment (FAB)

made the ionization and analysis of biomolecules possible. The FAB process utilizes a

matrix and an energetic beam of particles to desorb the sample mixture from a surface.

The FAB matrix is typically composed of the either m-nitrobenzyl alcohol or glycerol. A

beam of Xe neutral atoms or Cs+ ions then sputters the sample (with matrix) from the

surface. Once the sample has been ionized, the molecules enter the mass spectrometer

for analysis. Although the analysis ofbiomolecules is possible using FAB, the mass

range is limited to samples of less than 7000 Da. Additionally, some large protein

samples many not be ionized by this technique, limiting the range of peptides and

proteins that can be studied. The presence of the matrix ions in the resulting spectrum

can make interpretation difficult. Although FAB is more amenable to higher salt

concentrations than other ionization methods, the sensitivity (only nanomoles) and matrix

requirement make FAB less desirable for biological mass spectrometry.50,52'53









The introduction of matrix-assisted laser desorption/ionization (MALDI)

simultaneously by Hillenkamp and Karas and Tanaka in 1988 allowed for the more

routine ionization and analysis of many classes of biomolecules.54 In MALDI, the

peptide or protein mixture is typically co-crystallized with a UV absorbing matrix.

Sinapinic acid, 2,5-dihydroxybenzoic acid (DHB) and a-cyano-4-hydroxycinnamic acid

(a-CHCA) are commonly used as MALDI matrices. These matrices absorb the laser

energy used to desorb the sample from a surface. By absorbing the laser energy, the

matrix helps to minimize sample destruction and allows for the formation of the gas

phase molecules. Once the sample has been co-crystallized with the matrix, the mixture

is spotted onto a MALDI target and placed in the ionization region of the mass

spectrometer. Although many types of lasers can be used to irradiate the sample spot, a

simple nitrogen laser operating at 337 nm is most often utilized. The sample, with

matrix, is desorbed from the surface and ionized. The exact mechanism of the

desorption/ionization process is still very much in debate.9'48'50'54-57

Although the MALDI process typically produces singly charged ions (either

positive or negative ions), many large biomolecules can be analyzed by using the MALDI

source coupled to a TOF mass analyzers. The sensitivity of the MALDI process has been

reported to be in the femtomole range.50 This makes MALDI very appealing in the study

of peptides and proteins, where the amount of sample for analysis may be limited. The

tolerance of salts and the ability to analyze complex mixtures also make MALDI

appealing for the study of many biomolecules. However, a few disadvantages to the use

of MALDI in biological mass spectrometry do exist. The matrix is ionized along with the

sample. These matrix ions are then detected, causing the interpretation of the low mass









ions to be difficult due to the presence of the matrix ions in this region of the spectrum.

In addition, proteins can be fragmented during the ionization event, further complicating

the resulting spectra.48'50'5557

The analysis of peptides and proteins by MALDI is routine. However, the use of

electrospray ionization (ESI) is rapidly becoming more prevalent. Electrospray

ionization is utilized primarily due to the gentleness of the technique, i.e., parent ions are

formed with little or no fragmentation.58'59 The first electrospray experiments were

performed by Chapman in 1937. In these experiments, a mobility spectrum was

produced for a series of salt solutions.60 By the mid-1960s, Dole and co-workers had

utilized electrospray to produce negatively charged polymer ions that were detected by a

Faraday cage.61'62 During the mid-1980s, John Fenn and co-workers demonstrated the

practicality of using electrospray ionization in the mass analysis of biomolecules.58'59

These advances in the use of electrospray ionization have brought the study of biological

samples to the forefront of mass spectrometric analysis.

The ESI process involves the production of gas-phase ions directly from liquid

solutions. The peptide or protein mixture is typically dissolved in a solvent system

comprised of water, either acetonitrile or methanol, and a small volume of either acid or

base. Acetic acid and formic acid are commonly added to promote the formation of

positive ions while either sodium hydroxide or triethylamine (TEA) are added to the

sample solution to promote the formation of negative ions. Once the sample has been

dissolved in the appropriate solvent system, the entire mixture is loaded into a syringe

and pumped into the electrospray ionization source.50'51'56'63 The sample will enter the

mass spectrometer via a tip or needle. A voltage is applied to the needle, allowing the









solution to disperse into a spray of charged droplets. These charged droplets then enter

into the gas-phase by application of either a sheath gas or heat. As the size of the charged

droplets decreases due to the evaporation process, the charge density associated with each

droplet will increase. This increase in charge density creates ions that carry multiple

charges. As more and more like charges are produced, the Coulombic repulsion forces

become more pronounced. If the repulsion forces become large enough to exceed the

force of surface tension of the solvent system, a Taylor cone (Figure 1-4) is formed. The

ions can then be ejected from the Taylor cone and are directed into the mass

spectrometer.63-69









SOxidation "






increases as the amount of solvent evaporated during the oxidation/reduction
reaction increases. Figure adapted from reference 65.
High Voltage
Power Supply


Figure 1-4. Formation of the multiply charged droplets occurs due to evaporation of the
solvent from the sample mixture. The charge exhibited by the droplets
increases as the amount of solvent evaporated during the oxidation/reduction
reaction increases. Figure adapted from reference 65.

As stated previously, the phenomenon of multiple charging is a main advantage of

the ESI process. Because the mass spectrometer measures the mass-to-charge ratio of the

sample, ESI allows for the analysis of very large molecules by many types of mass

spectrometers. Electrospray ionization sources have been interfaced with TOF mass

spectrometers, quadrupole mass spectrometers and ion trap mass spectrometers. A










typical ESI mass spectrum can be seen in Figure 1-5. The molecular weight of the

sample can be calculated from the spectrum. The mass of the charge state shown in

Figure 1-5 can be multiplied by the respective charge to obtain the molecular mass of the

sample. While the mass of each sample remains the same, the m/z ratio can change,

depending upon the number of charges generated per sample.

Another advantage of the ESI process is the small sample volume requirement.

Sample consumption is low due to flow rates on the order of 300 nL/min (or lower) and

sensitivities in the picomole to high femtomole range.






zoom in





7790 7792 7794 7796 7798 7800 7802
700 750 800 850 900
m/z

0
t-
0
0
70

MW: 8559.8139 +11 charge state


Figure 1-5. ESI mass spectrum for ubiquitin. There are 3 different charge states seen for
this protein above. The +11 charge state has been deconvoluted to obtain the
molecular weight of the sample.

These sample requirements make ESI advantageous for the study of peptides and

proteins, where the total amount of sample available for analysis may be limited. In

addition, the electrospray ionization process can be achieved without the addition of the









complicated matrix as in MALDI and FAB. The electrospray source can also be

interfaced relatively easily to liquid chromatographs and capillary electrophoresis

instruments. This makes the application of hybrid mass spectrometric methods easy to

implement.50,51,56,66,67,70

Although the ESI process has several advantages that make the method appealing

for the study of biomolecules, several disadvantages of the technique do exist. The

presence of salts and buffers can significantly reduce the sensitivity of the electrospray

process. The presence of salt adducts can also complicate the interpretation of the

resulting spectrum. The overall signal intensity of the resulting charge states can also be

compromised. In addition, many biological samples may contain buffers that are not

suitable for the electrospray ionization process.50'51,56'66 A desalting procedure or

microdialysis is often required prior to the ESI event. The necessary sample preparation

techniques can make rapid analysis ofbiomolecules by ESI-MS difficult. Elimination of

the salts and buffers can be achieved by the use of off-line liquid chromatography or the

use of C18 spin columns. Microdialysis has also been reported for the clean-up of many

biomolecules. The necessary elimination of the salts and buffers common to many

biological samples results in spectra with higher S/N ratios that are much easier to

interpret. Complex mixture analysis is also difficult with the ESI process. The

formation of the multiply charged ions can be rather confusing, especially for these

mixtures. The sample purity must be questioned before the ESI process. If the sample is

relatively impure, the resulting spectra are difficult to interpret due to the signal

suppression associated with the production of the multiply charged ions. Another

disadvantage of the electrospray ionization process is the requirement that the spray









solution contain at least 50% organic content. The electrospray current and overall spray

can be compromised by the use of a solvent system comprised of 100% aqueous content.

The addition of the organic content to the spray solution makes the study of biomolecules

in solutions that mimic the natural environment of the cell impossible.50'51'56'66

Nanoelectrospray ionization (nanoESI), an emerging technique in the study of

biological samples, has recently been used to circumvent some of the disadvantages

associated with the traditional electrospray ionization process. In nanoESI, the traditional

spray needle is replaced with a small needle positioned very close to the entrance of the

mass spectrometer. The nanoESI process has shown an increased efficiency over

traditional ESI, allowing for further reduction of the amount of sample needed for each

analysis. In addition, the flow rate associated with nanoESI is at the very least an order

of magnitude lower than that of traditional ESI. The main advantage to using nanoESI

over traditional ESI is the salt tolerance. The droplets in nanoESI are much smaller in

size (-200 nm) than traditional ESI, making the amount of solvent that needs to be

evaporated lower. As a result, salts and buffers are less concentrated in the nanoESI

process. The resulting spectra will have higher S/N ratios, making the interpretation of

the spectra less difficult.50'76

Bottom-up versus Top-down Proteomics

As stated previously, mass spectrometry has emerged as a method of choice for the

study of proteins and peptides. However, two distinct mass spectrometric approaches are

currently utilized in these studies: bottom up proteomics and top-down proteomics. Each

of these approaches will be discussed below.

Bottom-up proteomics refers to the use of two-dimensional gel electrophoresis and

sample digestion combined with mass spectrometry to obtain the desired information









about the protein. Typically, the sample mixture is separated by isoelectric point and

mass during the electrophoretic event. Upon completion of the initial separation step, the

resulting protein bands are visualized by staining and excised from the gel. Either

chemical or enzymatic proteolysis is performed next. These procedures cut the protein

into smaller peptide fragments that are easier to analyze. Once the protein has been

cleaved into smaller peptides, two different mass spectrometric approaches can be

utilized. In peptide mass fingerprinting, the peptide digestion products are analyzed by

mass. The masses obtained are then used to identify the original protein. The second

approach utilizes tandem mass spectrometry (MS/MS) to obtain protein identification.

Although bottom-up proteomics is routinely used, there are a few drawbacks to this

approach. The prior isolation of the peptide fragments from the digestion procedure

make rapid, high-throughput analysis difficult. In addition, sequence information is not

normally obtained for the entire protein. Portions of the sequence may not be identified

by this approach, making complete localization of the modifications difficult. Sequence

coverage for the bottom-up approach typically ranges from 5-70%. The low resolution

achieved with this approach is also problematic.77-80

The disadvantages associated with the bottom-up approach can be circumvented by

the use of top-down proteomics. The top-down approach focuses on studying intact

protein samples without the need for prior fractionation steps. This approach often

reveals analytical targets such as post-translational modifications and biomarkers for

further identification. Top-down proteomics allows for 100% sequence coverage

(theoretically) by utilizing both collisional dissociation and electron capture dissociation

to obtain complementary fragment ion pairs. The resulting fragment ion pairs are then









used for database searching, confirmation of large stretches of the protein sequence and

localization of any modifications. Figure 1-6 illustrates the major differences between

the two approaches to protein identification. Although the use of top-down proteomics is

gaining popularity, the technique is not as widespread as the bottom-up approach. Most

top-down studies are performed using Fourier transform ion cyclotron resonance mass

spectrometry (FTICR-MS) coupled to an electrospray ionization source. The need for the

FTICR-MS instrumentation is the main drawback of the top-down approach. The

FTICR-MS systems are rather expensive and complex.


A)
Peptnde
ions from
proteolysis -- -
in solution
Bottom-up
Linear approach
protein ....
N C


B)
Intact Top-down
protein ion approach

Fragment
ions from -
gas-phase PTM
dissociation


Figure 1-6. A) Bottom-up proteomics requires digestion of the protein sample prior to
mass spectrometric analysis. B) The top-down approach allows for mass
spectrometric analysis of the intact protein sample. Figure adapted from
reference 80.

In addition, mixtures and complex protein samples are often difficult to analyze due

to the signal suppression often seen with the use of electrospray ionization. Complete

localization of modifications may also be difficult with the current MS/MS capabilities of









the mass spectrometers.22'80-83 The issues associated with the MS/MS capabilities will be

discussed in more detail in Chapter 2.

MARCKS Proteins

Of particular interest for this research is the use of mass spectrometry and tandem

mass spectrometry to study the phosphorylation of a small portion of the MARCKS

protein. MARCKS, myristoylated alanine-rich C kinase substrate, is an acidic protein

comprised of 309 amino acids (Figure 1-7).84 Protein myristoylation involves the

covalent attachment of myristic acid (n-tetradecanoic acid) to the N-terminal glycine

residue. This is an irreversible modification that promotes weak and reversible

protein-membrane and protein-protein interactions.85 The MARKCS protein is a known

cellular substrate for protein kinase C. The protein is believed to bind calmodulin in a

calcium dependent manner and to cross-link filamentous (F) actin. Located within the

MARCKS protein are three highly conserved domains. The effector domain is central to

the function of the MARCKS protein. This domain, also called the phosphorylation site

domain (PSD), is a basic peptide of 25 amino acids. This region contains five series that

are capable of phosphorylation. Phosphorylation of this domain may be crucial to the

overall function of the MARCKS protein. The PSD region is highlighted in red in Figure

1-7. The second domain is the myristoylated amino-terminal domain. This domain helps

to anchor the protein to the inner surface of plasma membranes. The MARCKS

homology domain, the final domain of the MARCKS protein, has no known

function.84,86-88

As stated above, the PSD region of the MARCKS protein appears to play a crucial

role in the overall function of the protein. The binding of calmodulin and F-actin

cross-linking occur in this region. Many previous studies have simply utilized the









function and behavior of the PSD region as an indicator of the overall function and

behavior of the MARCKS protein.

IMGAQFSKTAAKGEATAERPGEAAV
25ASSPSKANGQENGHVKVNGDASPA

49AAE P G AK E E L Q AN G S AP A A D KEEP
73A S G S AA T P AA EKD E AAAATEPGA
97GAAD K E AAEAEPAEP S S P AAEAEG
121A S A S S T S S P K AE D GA AP S P S S E T P

145K KKKKRF S F KK S F KL S G F S F K K S K
169K ESGEGAEAE GAT AE GAKDEAAAA

93A GGEGA A APGE Q A G GAGAE G A A G G
217EPREAEAAEPEQPEQPEQPAAEEP
241QAEEQSEAAGEKAEEPAPGATAGD
265A SSAAGPEQEAPAATDEAAASAAP
289AA S P E P Q P E C S P E A P P A P TAE

Figure 1-7. The sequence of the intact MARCKS protein. Protein myristoylation occurs
at the N-terminal glycine. The phosphorylation site domain, comprised of 25
amino acids, is highlighted in red.

A previous FAB-MS89 study indicated that series 1 and 2 are modified within the

PSD region. However, in that study, the PSD segment was comprised of 21 amino acids,

compared to the 25 amino acid segment studied in this dissertation. In addition, the

previous work focused on a PSD segment that contained only 4 series capable of

phosphorylation, as opposed to the 5 series present in the 25 amino acid sequence.89

Most studies do not involve the intact protein due to the difficulties associated with the

study of membrane proteins such as MARCKS. Identification and localization of any

modifications, specifically phosphorylation, in this region may provide for a better and

more complete understanding of the intact MARCKS protein.87'88,90









Overview

The next chapter is dedicated to a discussion of Fourier transform ion cyclotron

resonance mass spectrometry (FTICR-MS). A detailed description of the operating

principles and the use of FTICR for proteomic studies will be presented. An explanation

of the various tandem mass spectrometric methods used to localize protein modifications

will also be introduced. The use of electron capture dissociation (ECD) experiments will

be highlighted. Chapter 3 will discuss the implementation and optimization of an ECD

source on a FTICR-MS system. The use of both a filament and a dispenser cathode for

the ECD event will be explored. Examples of the ECD of both modified and unmodified

peptides will be presented. In Chapter 4, a study of a series of MARCKS analogues (test

peptides) by ECD-FTICR-MS will be presented. ECD results will be presented, along

with the relevant fragmentation summaries for these modified peptides. The relative

abundance of each of the respective fragment ions will also be discussed. The use of

ECD to study both the singly and doubly phosphorylated PSD region of the MARCKS

protein will be presented in Chapter 5. The final chapter, Chapter 6, will conclude this

work. This chapter will offer a summary of the previous chapters, as well as outlining

some future or proposed work.














CHAPTER 2
FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS
SPECTROMETRY

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), a mass

spectrometric technique that is continually evolving, has historical origins that can be

traced back to the early 1930s. During this time, E.O. Lawrence used the theory of ion

cyclotron motion to implement charged particle acceleration in cyclotrons. In these

experiments, protons were accelerated to high speeds, allowing for studies of atomic

nuclei.91-93 Twenty years after the initial cyclotron motion studies, Sommer and

colleagues developed the omegatron. This instrument was utilized to measure the

cyclotron resonance frequency of a proton, leading to the determination of the charge-to-

mass ratio of the proton.91'94'95

The next two decades saw the further development of ion cyclotron resonance

spectroscopy. The introduction of the ion cyclotron resonance spectrometer allowed for

the study of many ion-molecule reactions.91'96-99 The development of the trapped ion cell

by McIver in 1970 continued the ion cyclotron resonance spectroscopy evolution. The

trapped ion cell permitted the trapping of ions in the analyzer region for as long as 0.1

seconds. A trapping time of less than 2 milliseconds was seen for the original analyzer

cells. The trapped ion cell differed in three respects from the previous cells. The gaseous

ions were produced and detected in the same region of the instrument, the cell was

constructed with plates on both ends to confine the ions in the cell during the analysis and









the cell relied on pulsed modes of operation.91'100 These developments contributed

greatly to the evolution of modem ICR-MS techniques.

The introduction of Fourier transform (FT) methods to the operating principles of

ICR-MS by Marshall and Comisarow continued the development of the ICR-MS

technique as an analytical tool. All of the advantages of the standard ICR approach were

present, along with the added advantage of FT data acquisition.91'101'105 The

implementation of soft ionization methods, and respective ionization sources, to these

mass spectrometric studies has brought the use of FTICR-MS to the forefront of

biological mass spectrometry. The coupling of electrospray ionization (ESI) and matrix-

assisted laser desorption/ionization (MALDI) on the front end of many FTICR mass

spectrometers allowed the analysis of many classes of biomolecules, including peptides,

proteins, oligonucleotides and carbohydrates.50'77'79'105-108

The attention that the FTICR-MS technique is currently receiving can be directly

attributed to its ability to make mass measurements with a combination of accuracy and

resolution that is the highest among mass spectrometers. The ability of FTICR-MS to

obtain a variety of analytical and physical measurements also makes this technology very

versatile. The Fourier transform ion cyclotron resonance mass spectrometer has been

interfaced with most known ionization methods to obtain high resolution mass spectra.

The ability to perform hybrid mass spectrometric experiments such as HPLC-FTICR-MS

and CE-FTICR-MS makes the analysis of many biomolecules routine. The ability to

perform tandem mass spectrometry and to examine ion chemistry also makes these

particular types of mass spectrometry very appealing.78'105'106









This chapter will serve as an introduction to the FTICR-MS technique. Particular

attention will be focused on the use of FTICR-MS in the study of biomolecules,

specifically peptides and proteins. The basic principles of operation will be outlined first.

The ability to trap the ions for extended periods of time to perform tandem mass

spectrometric analyses will be highlighted. Particular attention will be paid to the

relatively new fragmentation technique known as electron capture dissociation.

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Instrumentation

The operating principles of most mass spectrometers rely on the spatial separation

of the ions in the presence of a magnetic or electric field followed by the subsequent

detection of the respective m/z values for the ions. In FTICR-MS, however, the mass

analysis and detection events are temporally separated. This temporal separation allows

the mass analysis and detection events to take place in one area of the instrument without

the need to separate and collect ions of different m/z values.50'109

Regardless of the application or field of study, all Fourier transform mass

spectrometers typically share four common instrumental components. The first

component is the magnet. The magnets used for Fourier transform experiments can be

permanent magnets, electromagnets or superconducting magnets. Permanent magnets

have low field strengths, limiting the performance of the FTICR-MS instrumentation. To

date, only a few mass spectrometers have been constructed with permanent magnets.

Electromagnets typically have field strengths of less than 2T, with 1T electromagnets

being the most common. The electromagnet FTICR-MS systems are often used to study

ions of relatively low mass-to charge ratios. However, as the size of the magnet and the

resulting magnetic field strengths increase, nine FTICR performance parameters increase

either linearly or quadratically.106'109 Figure 2-1 outlines the performance features that









increase with the increased field strength. Because of the desire to use the optimal

FTICR performance parameters, most FT mass spectrometers utilize superconducting

magnets. These magnets typically have field strengths of 3T to 9.4T, with 12T systems

becoming more prevalent.106 A 14.5T FTICR-MS system has recently been installed at

the National High Magnetic Field Laboratory (NHMFL).110 The size of the

superconducting magnet makes miniaturization of the FTICR-MS technique impossible.

The requirement that that magnet must be held at 4.2K also makes this technique rather

costly. The temperature is maintained by the use of both liquid helium and liquid

nitrogen, both of which must be replaced quite often. Despite these disadvantages,

FTICR-MS is still routinely used due to the increased mass accuracy and resolution when

compared to other types of mass spectrometers.91'106


Highest non-coalesced mass B) Trapped ion upper mass limit
Resolving power 25 T Ion energy 25
Axialization efficiency Ion trapping time
Scan speed for LC/MS Number of ions
2D-FT resolving power
9.4 T
7T
A7 9T
0 B 0(tesa) s ao (gtela)
0 25 0 25


Figure 2-1. FTICR-MS performance features that increase with an increase in the
magnetic field strength. A) Parameters that increase linearly with magnetic
strength. B) Performance parameters that increase quadratically with
magnetic field strength. Figure adapted from reference 105.

The second component of the FTICR-MS instrument is the trapping (analyzer) cell.

Most FTICR-MS experiments are performed in Penning traps. The Penning trap uses a

static magnetic field applied in the z-direction to confine the ions. In order to prevent the

ions from escaping through the rear of the cell, a three-dimensional axial quadrupolar

electrostatic potential is applied to each of the two end cap electrodes. The typical ICR









cell, composed of a pair of trapping electrodes and two pairs of electrodes to provide the

excitation and detection potentials, resides inside the bore of the superconducting magnet.

Almost any trapping cell geometry can be used as long as the necessary electrodes and

potentials are present.105'111,112

Two types of trapping cells are commonly used: the cubic cell and the open

cylindrical cell. In the cubic cell, six plates are arranged in the shape of a cube (Figure

2.2).


XD






x D




T T





Figure 2-2. Examples of a cubic trapping cell (top) and an open cylindrical trapping cell.
E denotes the excitation plates, D denotes the detection plates and T denotes
the trapping plates. Figure adapted from reference 105.

One pair of opposing plates is oriented orthogonal to the direction of the magnetic

field while two pairs of plates lie parallel to the field. The trapping plates lie

perpendicular to the field lines. Many cubic cells are comprised of trapping plates with

small openings to allow ions produced externally to enter the cell for excitation and

detection. The excitation and detection events occur with the remaining four plates of the









cubic cell. Because larger cells can trap more ions for analysis, the use of open

cylindrical cells has become more prevalent. The open cell (Figure 2-2) consists of six

electrodes that perform the same functions as the electrodes of the cubic cell. The two

cylinders at the ends of the cell serve as the trapping electrodes. The center cylinder is

divided into four electrodes that function as the excitation and detection plates. The

cylindrical shape of the open cell makes this particular geometry better suited for

placement into the bore of the magnet. Many current commercial and academic FTICR

mass spectrometers utilize the open cell geometry for experiments.106,109,113

An ultra-high vacuum system is the third component of all FTICR systems. For

optimal performance, and to achieve high resolution measurements, operating pressures

of 10-8 to 10-10 Torr are required. Although diffusion pumps have been used to achieve

these pressures, cryogenic pumps or turbomolecular pumps are used more often. Because

low pressure is only required for ion detection, other events in the experimental sequence,

such as ion formation, can be performed at elevated pressures. Typically, several stages

of pumping are used to ensure that the low pressure requirement is met once the ions

reach the cell region.106,109

The last component of the FTICR-MS instrument is the data system. The data

system is comprised of a frequency synthesizer, a delay pulse generator, a broadband r.f

amplifier and preamplifier, a fast transient digitizer and a computer. The computer must

be able to coordinate all the devices utilized during the data acquisition, as well as during

the data processing and analysis steps. As the computers used to control the instruments

and electronic components become more sophisticated, the amount of data that can be









collected and processed becomes larger.106,114 The ability to automate the acquisition

process also becomes a reality.

Ion Motion

Cyclotron Motion

The ion cyclotron motion exhibited by a circular orbiting ion is derived from the

interaction of the ion with a unidirectional magnetic field. An ion with a charge, q,

moving in the presence of an electric and magnetic field will experience a force that is

perpendicular to both the direction of the ion velocity and the magnetic field. The force

experienced by the ion can be calculated using Equation 2-1:

dv (2-1)
Force = ntm s acceleration = m- = qE+qv B.
dt

In Equation 2-1, m is the mass of the ion, q is the charge on the ion, v is the

velocity, E is the electric field and B is the strength of the magnetic field. The force

experienced by the ion, called the Lorentz force, causes the ions to travel in an orbit that

is perpendicular to the planes determined by both the velocity and the magnetic field.

Assuming that the ion velocity remains constant, and there are no collisions, the magnetic

field bends the ion path into a circle. As seen in Figure 2-3, positive and negative ions

will orbit in opposite directions within the magnetic field.

For the ion to experience circular motion, the Lorentz force must equal to the

centrifugal force. If the ion velocity in the xy plane, the plane that is perpendicular to B,

is denoted by vxy and the angular acceleration is denoted by dv/dt = v2r, Equation 2-1

becomes:

my2 (2-2)
= qv B.
r









Equation 2-2 can then be solved for the radius, r, of the cyclotron orbit:

(2-3)
my
r=
qB

Angular velocity, o, about the z-axis is defined by o = vxyr. Substituting this value of

angular velocity into Equation 2-2 gives:

mco2r = qBcor. (2-4)

Equation 2-4 then allows for the calculation of the cyclotron frequency, as seen by

Equation 2-5:


qB (2-5)
0)=
m

A simplified expression for the cyclotron frequency can be seen in Equation 2-6:


qB (2-6)


Equation 2-6 was obtained by substituting the relationship ) = 2Zit = 2finto Equation

2-5. A sample calculation for the cyclotron frequency of an ion of m/q 858 experiencing

a magnetic field for 4.7T can be seen below:

(2-7)
1.602 x 10 C 4.7T 83
f = =83.5kHz.
27r- 858u 1.673x10" kgu'

Several conclusions can be drawn from Equations 2-1 through 2-6. First, the

cyclotron frequency is determined by only three parameters: the magnetic field strength,

the charge on the ion, and the mass of the ion. The cyclotron frequency is also

independent of the velocity of the ion, and is therefore independent of the kinetic energy

of the ion. This means that all ions of the same m/q ratio will have the same cyclotron

frequency. Lastly, the frequencies calculated from Equation 2-6, at many representative









magnetic field strengths, lie in the kilohertz (kHz) to megahertz range (VMHz). This range

is readily detectable by most available instrument electronics.105,106



V V
+
C qv x B qv xB





Figure 2-3. The magnetic field bends the ions into a circular orbit as long as the ion
velocity remains constant and there are no collisions. The positive and
negative ions orbit in opposite directions. Figure adapted from reference 105.

Trapping Motion

Ion motion along the axis of the magnetic field, the z-axis, is unrestricted. If the

ion is moving parallel to the magnetic field, no force is experienced from the field. The

ions are only confined in the xy plane, allowing the ions to escape along the z-axis. By

applying a trapping voltage, Vtrap, to the end cap electrodes of the trapping cell, the ions

can be confined along the z-axis as well. The trapping voltage applied to the electrodes is

typically a three-dimensional axial quadrupolar potential.105'115 This potential can be seen

in Equation 2-8:

(2-8)
(D =V V +-- (2z'- r').
'"> "' 2a'

In Equation 2-8, Vtap, is the trapping voltage, r is the radial position of the ion in the xy

plane, a is a measure of the trap size and yand a are trap shape dependent constants.

Equation 2-8 can then be solved for the ion z-motion:

mdz (2-9)
F-.= =-qVcD(x, y,z).
dt'









This can then be used to determine an ion z-position that oscillates with time with a

frequency given by:


1 2qFa (2-10)
v =
2fIf ma'

Magnetron Motion

The combination of the magnetic and electric fields creates a three-dimensional

trap. This allows ions to be stored in the analyzer cell for extended periods of time.

However, the trapping motion and the cyclotron motion are not coupled. Although the

trapping and cyclotron motions are not coupled, these motions together introduce a third

type of ion motion: magnetron motion. The trapping potential of Equation 2-8 also

produces a radial force given by Equation 2-11:


qV a (2-11)
F =qE,= a2 r.
a

The force acts upon the ions in an outward direction that opposes the inward-

directed Lorentz force from the magnetic field. By combing Equations 2-5 and 2-10, an

equation for the ion motion subject to a static magnetic field and a three-dimensional

axial quadrupolar potential can be obtained:


SqV a (2-12)
F = moor = qB -'r r.
a

Solving Equation 2-12 for zero gives:


2 qBo qV ca (2-13)
S + "" = 0.
m ma

Equation 2-12 is a quadratic equation with respect to o). However, o) is

independent of the radius, r. Each ion motion frequency is then independent of the ion










position within the trap. By solving Equation 2-13 for wo, two natural rotational

frequencies are obtained. The first frequency, seen in Equation 2-14, is the unperturbed

cyclotron frequency that is observed in the absence of a d.c. trapping potential:

(2-14)
S=--+
2 2

The second frequency, Equation 2-15, is the magnetron frequency:

(2-15)
C 2 2 2
D =- --
S2 2 2

The three natural ion motion modes can be seen in Figure 2-4 Both the magnetron

and trapping frequencies are much less than the cyclotron frequency. Because of this,

these frequencies are usually not detected.105'106












x Vm Vc


Figure 2-4. Natural ion motion modes of an ion trapped in an ICR cell. The magnetic
field is aligned with the z-axis. The ion modes are trapping motion (VT),
cyclotron motion (v,) and magnetron motion (vm). Figure adapted from
reference 105.

Experimental Operation of FTICR-MS

The experimental events for FTICR-MS studies can occur in the same space. This

is in stark contrast to other types of mass spectrometry where the ionization, mass









analysis and ion detection take place in different parts of the mass spectrometer.50'105 A

typical FTMS experimental sequence can be seen in Figure 2-5. The events of a generic

sequence are as follows: quench, ionize, delayss, excite, detect, quench. The quench

pulse is the first event to occur. In this event, the analyzer cell is emptied of any ions that

may be present from previous experiments. The ions are typically ejected along the z-

axis. A quench pulse of approximately 1 millisecond is usually sufficient to empty the

cell of all unnecessary ions.












Time Deliys

Time


Figure 2-5. An example of a generic FTICR-MS experimental sequence. Time delays
can be added between the ionization and excitation events for ion-molecule
reactions or dissociation events.

Ionization, or the formation of the ions, typically occurs next. The ions can be

formed in one of two ways, either internally or externally. The ions that are formed

externally, such as those formed by ESI or MALDI, must be transported to the analyzer

cell. The ion transport can be accomplished by the use of ion optics or einzel lenses.

Figure 2-6 shows an example of the ion optics for a typical FTICR-MS instrument. A

series of delays can be utilized after the ionization event. The delays allow for the

introduction of collision gases, laser pulses or electrons into the analyzer cell for tandem









mass spectrometry.91'105'109 The use of FTICR instruments to perform tandem mass

spectrometry experiments will be discussed in more detail below. The use of the delay

also allows for ion-molecule reactions to take place prior to the excitation and detection

events.

Gate valve

HVO PL9
HVO
To source XDFL \ To cell
4- YDFL


PL1P4 FOCL FOCI2
V __ .

Figure 2-6. The ion optics utilized for ion transport from the source region to the
analyzer cell of a typical FTICR instrument manufactured by Bruker
Daltonics. PL1 through FOCL2 are a series of cylindrical optics or plates that
each have a voltage applied. The application of these voltages allows for the
transport of the ions.

Excitation is the next event in the FTICR-MS experiment. Typically, the ions that

are formed and trapped in the analyzer cell only have a small amount of kinetic energy.

Excitation is produced by applying a uniform electric field oscillating at or near the

cyclotron frequency of the ions of a particular m/z value. In FTICR experiments,

excitation can be utilized in three ways. Excitation is typically used to accelerate the ions

coherently to a larger, detectable orbital radius. In addition, excitation can increase the

ion kinetic energy above the threshold for ion dissociation or ion-molecule reactions or

can be use to accelerate the ions to a cyclotron radius that is larger than the radius of the

ion trap, effectively removing unwanted ions from the cell prior to dissociation or

detection.105'106 Figure 2-7 shows the uses of ion cyclotron excitation. All ions of the

same m/z value are excited coherently. The ions are grouped as tightly before and after

excitation, allowing ions of the same m/z to undergo cyclotron motion as a packet. The









periodic cyclotron motion of the ions then produces an image signal that can be

amplified, digitized and stored for later processing by a computer.106










Figure 2-7. Uses of ion cyclotron excitation can include: acceleration of the ions to form
an ion packet at a detectable radius (left), increasing the kinetic energy above
the threshold for dissociation (middle), and ejecting ions of a particular m/z.
Figure adapted from reference 105.

Detection is the final stage of the FTICR experimental sequence. The image

current that is produced following excitation provides FTICR-MS with some unique

capabilities over other types of mass spectrometers. All other mass spectrometers detect

ions by destructive means. With the image current detection, the ions are not destroyed

during the detection event. This allows the ions to remain in the cell after detection and

to be remeasured without the need to produce more ions. The ability to remeasure the

ions is an inherent advantage of the FTICR-MS technique. FTICR-MS can also be used

to detect ions of many masses simultaneously. Broadband detection can be achieved by

the application of many frequencies during the excitation event. This is typically

accomplished by using a r.f chirp. For example, a frequency synthesizer can sweep over

frequencies from 100 kHz to 10 MHz in a 1 millisecond time period, causing all ions

with cyclotron frequencies in this range to be excited. The image signal, or transient, that

results is comprised of different frequencies and amplitudes. By applying a Fourier

transform function to the time domain transient, the frequency components of the signal









can be obtained. The mass spectrum is then obtained by application of a calibration

formula derived from the cyclotron equation to the frequency spectrum (Figure 2-8).106,109

The last event of the experimental sequence is another quench pulse. This pulse

once again clears the cells of any unwanted ions. The entire experimental sequence can

then be repeated as many times as desired. The experimental sequences can vary in both

length and complexity, allowing for several different types of analysis. Each scan that is

collected can be signal averaged to obtain spectra with better signal/noise (S/N) ratios.

Such signal averaging improves the overall quality of the collected spectra.




Fourier transform




0 20000 40000 60000 80 000 1 20000 152000 560 5k 600 650 700 750 BOO B50
time m /

Figure 2-8. Example of the Fourier transform of the time domain transient to obtain the
mass spectrum for substance P.

Mass Resolution

The mass resolution achievable with FTICR-MS instrumentation is unparalleled.

Mass resolution (m2 mi > Amsi..,,) is defined as the point where a valley begins to

appear between peaks of equal height and shape that are separated by Ame..,, The term

Ame..,, is the full width of a peak at half maximum peak height. Mass resolving power is

defined at m/Ame..... The maximum number of mixture components that can be resolved

increases with mass resolving power. This allows identification of most mixture

components without the need for prior separation steps. High resolution can be achieved

in broadband (normal) mode or in heterodyne (narrow) mode. In the heterodyne mode,









the sampling occurs at a lower rate, allowing for a transient to be acquired, and thus

higher resolution for the sample.105,106

High mass resolution is achieved by recording a long time domain (transient)

signal. The number of data points that are collected, and that will comprise the resulting

transient, can be selected prior to the acquisition. However, there is a limit to the length

of the transient (number of data points) that can be collected and subsequently processed.

Currently, ~106 data points can be routinely collected and processed by modern computer

systems. The number of data points that are required to record a transient of desired

length depends upon the data sampling rate.106 This relationship can be seen in Equation

2-16:

N (2-16)
"q S

In Equation 2-16, Tis the transient duration, Nis the number of data points and S is

the sampling rate. The Nyquist criterion states that the sampling rate must be at least

twice the highest frequency that is recorded. The sampling rate is determined by the

lowest mass ion that is recorded during the acquisition. As seen by equation 2-6, the ion

of the lowest mass will have the highest frequency. The mass resolution will increase

with an increase in the number of data points collected.106 Figure 2-9 shows the effects

of collecting various numbers of data points on the resolution of the resulting spectra.

The highest mass resolution that can be attained for a particular data set is given by

Equation 2-17:


R fT (2-17)
2










In Equation 2-17, R is the resolving power, f is the cyclotron frequency, and Tis the

transient duration. Better resolution is obtained by acquiring a longer transient.

However, there is a limitation to the length of the transient that can be acquired. The

transient signal amplitude decays over time as collisions between ions and neutrals

destroy the coherent ion packet within the analyzer cell. Because of this, FTICR-MS

experiments are carried out with ultra-high vacuum to minimize the resulting collision

frequency. Space charge effects can also affect the resolution. As more and more ions of

the same charge reside in the cell, the likelihood of Coulombic repulsion increases. The

accuracy and resolution of the experiment can be compromised by the space charge

effects.106,116,117




128K


*^|T-*T T-WT^1BBW*




256K



0 00 00 000 000 00000 0000 856 5 857 0 857 5 858 0 858 5 859 0 859 5

i ,i


512K


0 100000 200000 0 ooooo 400o00 50 0000 856 5 857 0 857 5 858 0 858 5 859 0
Time m/z

Figure 2-9. Effect of collecting more data points on the resolution of the resulting
spectra. The resolution for the mass analysis ofubiquitin increased as the
number of data points collected was increased.


AL"

:: -kid^T^^w'^T'^^'^^









Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass
Spectrometry

An inherent advantage to the use of Fourier transform mass spectrometry in the

study of biomolecules is the ability to perform tandem mass spectrometric measurements.

In tandem mass spectrometry, the precursor ion is activated and dissociated. Mass

analysis of the resulting product (secondary) ions follows. Tandem mass spectrometry

experiments, while more complex than regular mass spectrometric experiments, can be

performed by simply altering the experimental sequence. In order to perform the tandem

mass spectrometry measurements with magnetic sector or quadrupole mass

spectrometers, additional mass analyzers are needed. With FTICR-MS, the experimental

sequence is amended to include pulse events for the selection of the precursor ion for

dissociation. As stated previously, the dissociation experiment can be accomplished by

the use of collision, lasers or electrons.105,106'118 The roles of these types of dissociation

will be discussed below.

Slow Heating Dissociation Techniques

Collisional dissociation involves the trapping of the ions in the analyzer cell prior

to dissociation. Once the ions are trapped, the desired precursor ion is mass selected. By

the application of an excitation pulse, all ions of higher and lower mass than the

previously selected precursor ion are ejected from the analyzer cell. Upon ejection of all

unwanted ions, the precursor ion is driven to an orbit of larger radius to increase the

kinetic energy of the ion.106,119 Equation 2-18 shows the relationship between the kinetic

energy and ion radius:


E q2B2r (2-18)
2m









The mass selected, kinetically excited ion can then undergo collisions with a gas

(typically Ar) admitted into the cell via a pulsed valve.120 The ions are not typically lost

from the analyzer cell assuming the pulse of gas pressure is not too high.

One disadvantage of the traditional collision induced dissociation technique is that

the product ions are formed away from the center of the cell. If the ions are not centered

within the analyzer cell, a decrease in the detection efficiency and resolution can result.

The use of a specific type of CID, SORI-CID, can overcome this disadvantage.121-123

Sustained off-resonance irradiation (SORI) uses a low amplitude r.f. pulse. The r.f pulse

is applied slightly above or below the resonant frequency of the precursor ion. This pulse

causes the ion kinetic energy to oscillate with time. The excitation frequency causes the

cyclotron orbit to expand and shrink repeatedly. The amplitude of the excitation is

traditionally low, leaving the ions in the center of the cell. The pressure is then raised

within the cell by the introduction of the collision gas. The ion can then undergo many

low-energy collisions that will slowly activate the ion until the threshold for dissociation

is met. The product ions are then formed near the center of the cell and are detected

efficiently.121-124 Figure 2-10 shows the continuous excitation experienced by the ions

during the SORI-CID event. Multiple excitation collisional activation (MECA) and very

low energy collisional activation (VLE-CA) have also been used to keep the ions near the

center of the cell for detection. However, for the dissociation of peptides and proteins,

the SORI-CID method is most often utilized. Disadvantages of the SORI-CID technique

include the low S/N for the resulting fragment ions and the time required for the

dissociation (>2 seconds/spectrum).105,109

















Time


Figure 2-10. Evolution of the ion cyclotron radius and ion trajectory during SORI-CID.
The ions of a previously selected m/z are alternately excited and de-excited
due to differences in the excitation frequency and the ion cyclotron frequency.
The ion packet should remain near the center of the cell during this event.
Figure adapted from reference 109.

Figure 2-11 shows the production of the product ions from collisional activation of

a peptide molecule. CID fragmentation breaks the amide bond to produce both N-

terminal and C-terminal fragment ions. The b (N-terminal) and y (C-terminal) ions can

be used to sequence many peptides and proteins.

R1 0 R3 0 H H
H2N I,. N --HOH
0 R2 0 R4


Path b/y


Ri H
H2N tY R2


b-ion


Figure 2-11.


R3 H
H2N r N OH
0 R4

y-ion


Cleavage of the amide bond during the slow heating dissociation techniques
results in the formation of N-terminal (b ions) and C-terminal (y ions)
fragment ions. Figure adapted from reference 118.


In addition to the production of the b and y fragments, loss of small neutrals such as

water and ammonia can occur. However, the use of CID for the localization of









post-translational modifications is more challenging. Typically, CID imparts large

amounts of energy into the peptide or protein. If cleavage occurs via the lowest energy

fragmentation pathway, there is high potential for loss of the labile modification prior to

dissociation. The loss of the modification can result in incomplete sequencing of the site

of modification. The resulting spectra will contain fragment ions indicating the loss of

the modification, making localization of the site of modification challenging.118

Infrared multiphoton dissociation (IRMPD) and blackbody infrared dissociation

(BIRD) have also been used in the study of peptides and proteins. In IRMPD, the

precursor ion is subjected to IR irradiation from a CO2 laser typically operating at 10.6

rm. The ions are slowly heated until dissociation begins. IRMPD also results in the

cleavage of the amide bond but has more internal fragmentation compared to CID.125-130

In the BIRD technique, the precursor ion is activated through absorption of blackbody

infrared photons produced by heating the vacuum chamber surrounding the analyzer cell.

Although the BIRD technique gives similar fragment ions to both CID and IRMPD,

BIRD is not typically used for peptide or protein sequencing. BIRD is most often utilized

to obtain the activation energies for the cleavage of both covalent and non-covalent

bonds.131-134

Electron Capture Dissociation

An exciting alternative to the collisional activation of peptides and proteins is the

use of electron capture dissociation (ECD). Developed in 1998 by Fred McLafferty and

colleagues, ECD has garnered considerable interest from the FTICR-MS community.135

This has been primarily due to the ability of the technique to not only sequence

polypeptides but to completely characterize and localize post-translational modifications.

ECD typically involves the use of low energy electrons to irradiate the precursor ion.









The precursor ion must be at least doubly charged, as the electron capture of a singly

charged species would result in neutral fragments that could not be detected by the mass

spectrometer. The ability to produce multiply charged ions by electrospray ionization

makes the coupling of ECD with ESI-FTICR-MS very desirable. Currently, ECD studies

are limited to FTICR-MS instruments due to the need to trap the ions in the analyzer cell

for extended periods of time.136-141 ECD has been attempted on a quadrupole ion trap

with limited success.142

The initial source of electrons was a conventional heated El filament mounted on

the rear axis of the analyzer cell. The rate of ECD for these sources was relatively low.

The ions had to be irradiated between 3 and 30 seconds, making the entire process very

time consuming. The time necessary for the irradiation event limited the coupling of the

ECD source with on-line chromatography. Typical HPLC peaks only last a few seconds,

all but eliminating the possibility of using ECD to fragment the HPLC separated

fractions. The optimal fragmentation occurs when the ion packet overlaps with the

electron cloud. If the trapped ions are not near the center of the cell, sufficient overlap

with the tightly focused electron beam cannot occur. In order to trap the ions in the

center of the cell, a pulse of Ar gas is commonly used. The gas cools and relaxes the ions

to the center of the cell. This allows for sufficient overlap of the ion packet with the

electrons. This process, however, adds additional time to the experimental sequence.

The insufficient overlap of the ions with the electrons and the low ion flux associated

with the El filament contributed directly to the long irradiation times associated with the

initial ECD studies.135,143-145









In 2001, Zubarev and co-workers replaced the standard El filament with an

indirectly heated dispenser cathode. The emitting area of the cathode is much larger,

increasing the overlap between the electron beam and the ion packet. The dispenser

cathode typically produces more electrons in a shorter period of time (higher flux). The

electrons also have more uniform kinetic energy. The irradiation time for the ECD event

using the cathode has been reported in the millisecond range. However, the irradiation

step is still the rate limiting step of the experimental sequence. The coupling of ECD

with both HPLC and CE separation prior to FTICR-MS detection has met with marginal
146-150
success.

Unlike the previously discussed slow-heating techniques, ECD is a nonergodic

process, i.e., there is little or no reorganization of energy throughout the peptide.135 The

fragmentation process occurs prior to the energy randomization. While CID

fragmentation is the result of amide bond cleavage, ECD results in the cleavage of the

amine bond.118 The N-terminal (c ions) and C-terminal (z ions) fragment ions can be

seen in Figure 2-12. The exact mechanism of dissociation is still very much in debate.

However, there are no favored sites of cleavage within peptides or proteins. The only

amino acid that does not exhibit cleavage by ECD is proline. This is most likely due to

the cyclic nature of the proline side chain. While the formation of the c and z ions is the

primary fragmentation pathway for ECD, a secondary pathway does exist. Figure 2-13

shows the formation of the less prominent a andy ions. This fragmentation pathway is

minor but can be seen for a large number of peptides.118










2HH H
R 1 0 N H 0 R H' HN-_NH
SPath c/z 3 0 R2

0 R2 O R4 O R4 OH

z-ion H2N N NH
0 R2
c-ion

Figure 2-12. The cleavage of the amine bond during electron capture dissociation to
form c and z product ions. The exact mechanism of this cleavage is not
completely understood. Figure adapted from reference 118.

The extent of cleavage by ECD varies from sample to sample. In the original ECD

study, the ECD of ubiquitin (-8.6 kDa) resulted in the cleavage of 50 out of a possible 75

backbone positions (67% cleavage) while the ECD of cytochrome C (-12.4 kDa) resulted

in the cleavage of 63 out of 103 backbone bonds (61% cleavage).135 Most early ECD

studies indicate that the extent of cleavage is greatest for smaller peptides or proteins

(<20 kDa). The lack of fragment ions for larger proteins may be due to the presence of

non-covalent interactions. The electron capture process will cleave the backbone bonds

but does not disrupt the non-covalent interactions, resulting in fewer fragment ions.118

The use of activated ion (AI) ECD has recently been used to circumvent the issues

associated with the dissociation of larger proteins. In AI ECD the ions are heated by

collisional activation or infrared irradiation while the electron capture process is

occurring. This effectively destroys the higher order protein structure, allowing for

fragmentation. As an example, conventional ECD of carbonic anhydrase (29 kDa)

resulted in no backbone cleavages while the AI ECD of the sample gave cleavage at 116

out of a possible 258 sites (45% cleaved).151










RIH R3 OH 2H Path a/y 1 H I R3 H
N N N
H2N N OH2N + C H2Nr OH
O R2 O R4 R2 O R4
a-ion y-ion

Figure 2-13. Formation of a andy products during the ECD event can also occur. This
fragmentation pathway is minor but is often seen for small peptides and
proteins. Figure adapted from reference 118.

ECD has gained considerable popularity in the field of biological mass

spectrometry due to the tendency of the fragment ions to retain the labile

post-translational modifications. This is in contrast to the slow heating techniques, where

the dominant fragmentation pathway is cleavage at the site of modification. Several

studies have involved localization of the site of sulfanation, phosphorylation,

glycosylation, acylation and methionine oxidation. In each case, the site or sites of

modification were correctly identified. The use of ECD in top down proteomics is

currently being exploited. In these studies, the intact, modified protein is fragmented by

ECD in order to determine the exact site of modification.152-157

ECD is also unique among other dissociation techniques for the ability to cleave

disulfide bonds (Figure 2-14) and to produce side chain cleavages. CID results in little

disulfide bond cleavage. The cleavage that does occur often results in the production of

overlapping b/y ions that are difficult to assign. The reduction and alkylation of the

disulfide bond prior to mass spectrometry is commonly done to alleviate these issues.

However, this process is time consuming and does not need to be performed prior to

ECD. Amino acid side chain cleavage also occurs with ECD. These cleavages, seen

most often for arginine, histidine, asparagine, methionine and lysine residues, result in the

formation of major ECD products.118,158,159









2H H

S-S \ RI-SH + S-R2
R2 --

Figure 2-14. The cleavage of the disulfide bond can occur during the electron capture
dissociation process. This is unique to ECD, as the cleavage of this bond
during the slow heating dissociation techniques occurs with very little
efficiency. Figure adapted from reference 118.

Conclusions

In the last 30 years, the field of Fourier transform ion cyclotron resonance mass

spectrometry has evolved from an academic research endeavor into a primary research

tool in the biological world. The ability to obtain mass measurements with both high

resolution and mass accuracy makes FTICR-MS appealing for the study of many classes

of biomolecules. The incorporation of electron capture dissociation into the FTICR

instrumentation has proved to be invaluable to the study of many post-translationally

modified peptides. The continued development of FTICR instrumentation and the

implementation of this instrumentation into the field of biological mass spectrometry will

transform the technique into the method of choice for the study of many biomolecules.














CHAPTER 3
IMPLEMENTATION OF AN ELECTRON CAPTURE DISSOCIATION SOURCE

Introduction

Electrospray ionization (ESI) coupled to Fourier transform ion cyclotron resonance

mass spectrometry (FTICR-MS) has become a primary analysis tool for many

biomolecules, including peptides and proteins. ESI-FTICR mass spectrometry is often

utilized for biomolecule analysis due to its gentle nature, ease of remeasurement, high

mass accuracy and high mass resolving power. Mass spectrometric fragmentation

methods are used to sequence many biomolecules, potentially allowing for the

localization of the sites of modification. Fragmentation method development is

continually evolving, as the sample mixtures become larger in mass while their mass

differences become smaller.105'106,109,160-162

The most commonly utilized mass spectrometric fragmentation method to identify

sites of modification is collision induced dissociation (CID). Typically, CID imparts

large amounts of energy into the biomolecule. This often results in the loss of the

modification prior to the cleavage of the amide bonds. Loss of the modification causes

incomplete sequencing and may not allow for complete localization of the modification.

Due to the desire to completely and accurately sequence proteins and peptides, a

complementary fragmentation method must be utilized.105'106'119

Electron capture dissociation (ECD) has proven to be a valuable complementary

fragmentation method to CID. Developed by McLafferty and co-workers138'163, ECD

utilizes low energy electrons for fragmentation. The electrons are produced by either a









standard rhodium or rhenium electron ionization filament or an indirectly heated

dispenser cathode. The low energy electrons produced from these sources enter the

analyzer region of an FTICR mass spectrometer (most commonly) and overlap with the

ion packet, inducing fragmentation. Because ECD is a nonergodic process, i.e., there is

little or no reorganization of energy throughout the protein or peptide, the PTMs remain

intact. This theoretically allows for more complete sequencing and localization of the

modifications. There are a few drawbacks to the use of ECD, however. If the ion packet

is not tightly focused in the center of the analyzer cell, sufficient overlap of the tightly

focused electron beam and the ion packet will not occur. This overlap is crucial to the

ECD fragmentation process. In addition, the long irradiation times (-1-30 seconds)

required for sufficient electron production and overlap currently limit the use of ECD to

Fourier transform ion cyclotron resonance mass spectrometers.40'118'135-138,140,152,163

Typically, CID and ECD produce complementary sets of fragment ions. In CID,

the amide bond is broken, resulting in the production of b/y ion pairs. ECD cleaves the

amine bond, resulting in the production of c/z ion pairs. In addition to the major ECD

fragment ions (c/z), the less predominant a andy ions are also produced. Secondary

fragmentation of the peptide backbone or loss of an amino acid side chain can also occur

during the ECD process.118,158,159 The major ECD fragment ions are illustrated in Figure

3-1.

In the work reported in this chapter, an ECD source was installed and optimized on

an existing FTICR-MS system. A series of modified and unmodified peptides and

proteins were studied by CID and ECD. Comparisons of fragmentation efficiencies when

using an electron ionization filament versus a dispenser cathode were made. In addition,










sustained off-resonance irradiation (SORI)-CID and ECD were utilized to obtain

maximum fragmentation and sequence coverage for various peptides and proteins. Once

optimized, ECD was utilized to localize the sites of modification for three different singly

phosphorylated peptide molecules.

cl c2 c3 c4
O O O O O

H2N-CH-C-N- -CH-C- -CH-C-N- -CH-C- C -OH
IH I H H H H
R1 R2 R3 R4 R5
z4 z3 z2 zl

Figure 3-1. Representation of the fragment ions formed during the ECD process. The
amine bond is cleaved, resulting in the retention of any post-translational
modifications.

Experimental Methods

Sample Preparation

The peptide and protein standards were purchase from Sigma (St. Louis, MO). The

phosphopeptides KIGDFGMTRDIYETDpYYRKGGK (pY denotes phosphotyrosine),

GnLAGPnLQSpTPLNGARR (nL and pT denote norleucine and phosphothreonine,

respectively) and SNKSQKLLRpSPRKPTRKISK (pS denotes phosphoserine) were

synthesized at Abbott Laboratories. These phosphopeptides were synthesized to mimic

commercially available peptides.164-166

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

All samples were analyzed on a Finnigan NewStar T70 (7.0T) Fourier transform

ion cyclotron resonance mass spectrometer (ThermoFinnigan). An instrumental

schematic can be seen in Figure 3-2. All experiments were performed in the analyzer cell

rather than the source cell region of the mass spectrometer. Ionization was achieved by

the use of an electrospray ionization source fitted with a heated metal capillary.










The peptides and proteins were electrosprayed in positive ion mode at a flow rate of

180 nL/min using a laser-pulled fused silica microspray tip. Final solution concentrations

were approximately 0.1 mg/mL in 1:1 acetonitrile:water. The addition of 2% acetic acid

(by volume) promoted the formation of positively charged ions.



Analyzer Cell Syringe

Source Cell -
^ I __, I 111111D I-` /l Ion Transfer Region
I II 7-Tesia /
ECD Source Superconductin
\ Magnet

E - -- -
Ultrasource

S, ESI Source

A I



TurboPump
Diffusion Pumps


Figure 3-2. Instrumental schematic of the Finnigan NewStar T70 Fourier transform mass
spectrometer. The dual cell is shown above. The source cell and analyzer cell
are separated by a small conductance limit. The source cell was utilized for
internal MALDI experiments while the analyzer cell was utilized for all other
experiments. The ions were transported from the electrospray ionization
source to the analyzer cell via a series of lens stacks located in the ion transfer
region.

The heated metal capillary was held at a temperature of 200 C. The electrospray

voltage was 2000V for all experiments. The positively charged ions were accumulated

for 1 second in an external hexapole prior to transfer to the analyzer cell. All ECD

spectra were acquired in positive ion mode, with 200 single scan acquisitions average for

each tandem mass spectrum. 512K data points were collected and processed.









Sustained Off-Resonance Irradiation Collision Induced Dissociation (SORI-CID)

SORI-CID was performed using a frequency offset of 1.5 kHz. The frequency for

the most intense (abundant) charge state for fragmentation was determined prior to

collisional dissociation. A SWIFT (stored waveform inverse Fourier transform) function

was applied, allowing for the isolation of the desired charge state for fragmentation. All

other ions were ejected from the analyzer cell. A pulse of argon (Ar) gas was introduced

into the analyzer cell. The collisions between the ion packet and the collision gas

allowed for the fragmentation of the sample. The sustained off-resonance irradiation

event, corresponding to the previously determine frequency offset, occurred next. This

was followed by a delay to allow the gas to be pumped away prior to detection.

Electron Capture Dissociation (ECD)

The first set of electron capture dissociation experiments was performed using a

standard rhenium electron ionization filament. The ECD source was mounted on the rear

axis of the source cell. The tightly focused electrons traveled through the conductance

limit located between the analyzer and source cells. The ECD event occurred in the

analyzer cell. The electron irradiation time was 10 seconds, with an electron energy of

2.2 eV (with respect to ground) and a current of 2.3 A. The electron emission current

was 0.7 iA. A pulse of Ar gas was introduced into the cell to relax the ion packet to the

center of the cell. This helped to produce sufficient overlap between the ions and the

electrons.

A series of ECD experiments were also carried out using an indirectly heated

dispenser cathode. The ECD experiments were performed using a 10mm

barium/tungsten dispenser cathode (HeatWave, Watsonville, CA) mounted on the rear

axis of the source cell. The ECD source operated at a potential of 5V versus ground









during the ECD event. The current was 1.7 A. The source trapping plate was held at

3.2V. The bias reflects the energy of the electrons entering the cell. The energy of the

electrons can be controlled by adjusting this bias. The electron irradiation time was 5

seconds. The emission current of the cathode was not known. A pulse of Ar gas was

utilized to cool the ion packet to the center of the analyzer cell for fragmentation.

Data Analysis

Data analysis was achieved using the MIDAS (Modular ICR Data Acquisition

System) data analysis software developed at the National High Magnetic Field

Laboratory (NHMFL).114,167 Each spectrum was baseline corrected and zero filled twice.

Hanning apodization was utilized during the data processing step.168 The THRASH

(Thorough High Resolution Analysis of Spectra by Horn)169 program was used to

generate the experimental peak lists obtained for both the SORI-CID and ECD

experiments. The monoisotopic masses obtained for each fragment ion were imported

into the ProSight PTM program (Kelleher group, UIUC) for generation of the

fragmentation summaries for each protein or peptide.170'171 The ProSight program was

utilized to positively identify the c/z and b/y ion pairs formed during the fragmentation

processes.

Results and Discussion

Upon installation of the original ECD source (El filament) on the Finnigan

NewStar FTICR-MS system, the electron energy was optimized. In addition, the electron

irradiation time was adjusted as to accommodate the pulse of Ar gas required for

sufficient overlap of the electron beam and the ion packet. The Ar gas pulse, when

introduced, increased the overall pressure in the analyzer cell. Additional time was

required for the ions to be relaxed to the center of the cell, increasing the total time of the










ECD slice of the experimental script. Figure 3-3 shows the ECD spectrum obtained for

the small protein ubiquitin (-8.5 kDa). In this case, the +10 charge state produced by the

electrospray ionization of the 0.1 mg/mL spray solution was isolated by the use of a

SWIFT waveform. All other charge states (and ions) were ejected from the analyzer cell

prior to fragmentation.



[M + 1OH]1

M QF V K T L T G KTI T L EVLE PS D TI ELNLV KLA
K I Q D KEG I P PD Q Q R LIFAG K Q L ED G RT L
'S DIY N IQ K EST L H LVL R L R GG






[M + 10H + e]






700 800 900 1000 1100
m/z



Figure 3-3. ECD mass spectrum obtained for ubiquitin using the El filament as the
electron source. The charge state isolated for fragmentation and the charge
state representing the capture of an electron are labeled above. The resulting
fragmentation summary is also shown above (inset).

The optimal electron energy for the electron capture dissociation of ubiquitin was

determined to be 2.2 eV. For ubiquitin, 11 out of a total of 75 backbone bonds were

cleaved. An increase in the total number of backbone bonds cleaved was expected when

the electron ionization filament was replaced with an indirectly heated dispenser cathode.

As stated previously, the dispenser cathode produces a higher flux of electrons than the









filament, allowing for more efficient overlap of the ion packet and the electrons. It

should be noted that adjusting the electron energy of the filament (higher or lower) did

not result in an increase in the number of backbone bonds that were cleaved.

Implementation of a Dispenser Cathode for ECD Experiments

In the fall of 2003, the standard El filament was replaced with an indirectly heated

dispenser cathode. The dispenser cathode, from HeatWave Labs172, is a standard

barium/tungsten cathode (Figure 3-4). Typically, the cathode operates at temperatures

ranging from 950-1200 C. As the operating temperature of the cathode is increased, the

voltage and wattage associated with the operation also increase. Under normal operating

conditions, the dispenser cathode provides greater than 10,000 hours of operation.

emitter face








heater power support legs
leads


Figure 3-4. Schematic representation of the dispenser cathode utilized for the ECD
experiments. The cathode is mounted on the rear axis of the analyzer cell.
Figure adapted from reference 170.

As with the filament, the electron energy associated with the cathode was

optimized. The initial energy utilized was 2.2 eV, the optimal energy for the filament.

However, at this energy, no electron capture was observed. Only the precursor ion was

present. Raising the electron energy did not result in fragmentation. However, when the

electron energy was lowered to 1.8 eV, the appearance of fragment ions was observed.










The first successful ECD spectrum obtained with the dispenser cathode can be seen in

Figure 3-5. In this spectrum, 33 total backbone bonds were cleaved (41 fragment ions

total). As with the previous ubiquitin spectrum, the +10 charge state of ubiquitin was

isolated and fragmented. The increase in backbone bond cleavages is due to using the

dispenser cathode rather than the filament.


[M + 10H]0











[M + 10H + e]




600 700 800 900 1000 1100 1200 1300
m/z


Figure 3-5. First ECD mass spectrum obtained using the indirectly heated dispenser
cathode. The resulting fragmentation summary is seen above. A total of 41
fragment ions were produced during the dissociation event. There is a
noticeable increase in the number of fragment ions obtained when compared
to the ECD of ubiquitin using the El filament.

Lowering the electron energy below 1.8 eV resulted in a decrease in the total

number of fragment ions that were produced. Although there was electron capture at the

lower energy, fewer fragment ions were formed, preventing maximum sequence

coverage. Therefore, all future ECD experiments involving the dispenser cathode

utilized an electron energy of 1.8 eV.










Comparison of the Electron Ionization Filament and the Dispenser Cathode

As stated in Chapter 2, the extent of cleavage for the ECD experiments performed

with an El filament is greatest for smaller proteins and peptides. Examples of this can be

seen in Figures 3-3 and 3-5. Traditionally, larger proteins are digested into smaller

peptides for ECD using an El filament. However, the use of the dispenser cathode for the

ECD experiments allows for the fragmentation of larger intact proteins. This is most

likely the result of the higher flux of electrons produced by the cathode.

Figure 3-6 shows the ECD spectra obtained for horse heart myoglobin (-17 kDa)

using both a standard filament and a cathode. For the ECD of myoglobin with the

filament, no charge state isolation was utilized.




A) filament




300 400 500 600 700 800 900
m /z
B) cathode






750 800 850 900 950
m /z


Figure 3-6. ECD mass spectra for horse heart myoglobin. A) Mass spectrum generated
with an El filament as the electron source. B) Spectrum obtained using a
dispenser cathode.

All charge states present after the initial electrospray ionization and mass analysis

were subjected to the electron beam. With the filament, a total of 9 fragment ions were

produced during the dissociation event. This resulted in -6% backbone bond cleavage.









For the ECD using the dispenser cathode, the +21 charge state was isolated prior to

fragmentation. When using the cathode, a total of 34 fragment ions were produced,

giving -22% backbone bond cleavage. The resulting fragmentation summaries for the

ECD experiments involving myoglobin can be seen in Figure 3-7. In addition, the

electron irradiation time for the filament was approximately twice as long as that for the

dispenser cathode.

G LSDG EIWQQVLNVQG KVEAN IAGH 1G LSDGEWQQVLNVQG KVEANIAG H
2 EGQEVLI R LFTG H PETLEK FDK FK H LK 2GQEV LI R LFTG H P ET LJE1K FDIK FKH LK
"T EA EM KA SE LK K H GTVVLTALGG I K5TE A SKED L KKHG GTVVL T A L GGI
"7LKKKGH HEAELKPLAQS HATKHlKP I 76LKK KG H H E ELK PLA QS HATKH KIIPI
102KYL EFISDAIIHVLH SK H PG DFGADA 102K Y LLE F IS D A I I H V L H SK H PG D FG A DA
128QGAMTKALELFR NDIAAKYKELGFQl 128QGAMTKA L ELFRN D IAAKYKELGFQ
1-aG 153G

Figure 3-7. Fragmentation summaries for the ECD of horse heart myoglobin. The
summary on the right was obtained using the cathode. The summary on the
left was generated using the El filament. More fragment ions were produced
with the cathode, allowing for greater sequence coverage.

Comparison of ECD and SORI-CID

The fragmentation summaries or patterns obtained from CID and ECD alone do not

typically provide maximum sequence coverage. However, when used together, the

fragmentation results allow for more complete sequencing of the peptide or protein.

Figure 3-8 shows the SORI-CID mass spectrum obtained for ubiquitin. The collisional

dissociation event was initiated by the introduction of Ar gas into the analyzer cell. A

total of 16 b ory-type ions were formed for the SORI-CID event. They resulted from

cleavage of 12 backbone bonds and gave -21% backbone bond cleavage, as can be seen

in the fragmentation summary in Figure 3-8.









As seen in Figure 3-5, 33 backbone bonds were cleaved for the ECD of ubiquitin,

giving 44% backbone bond cleavage.


[M + 10H]


M Q I F V KT L T G KT IT L E LEI S D T I E N V KA
"KI Q DKEGIPPDQQRL AGK Q L E DG GRTL
"S DY NI Q ELS TL HLVLRLRG G


600 700 800 900 1000 1100 1200
m/z


Figure 3-8.


SORI-CID spectrum obtained for the dissociation of ubiquitin. Sixteen
fragment ions were produced during the dissociation event. The resulting
fragmentation summary can be seen above.


When the fragmentation summaries for the SORI-CID and ECD of ubiquitin are

combined, a total of 39 (out of a possible 75) backbone bonds were cleaved, resulting in

52% backbone bond cleavage. The combined CID/ECD fragmentation summary can be

seen in Figure 3-9.

1MQ[ FV|KTLTGKTITLE|V; E LPSDTIENVLKA


29K IQDKEG IP PD Q QRKL FAGK 7 QLEJDG RT7L


67S DYl QK S TLHL LRL RGIG



Figure 3-9. Combined ECD (red) and SORI-CID (blue) fragmentation summary for
horse heart myoglobin. By combining the summaries, greater than 50%
backbone bond cleavage was obtained.










Electron Capture Dissociation of Modified Peptides

Although the ECD source was successfully utilized for the fragmentation of peptide

and protein standards, questions remained as to whether the site of modification for a

post-translationally modified peptide could be localized. In order to determine this, three

singly modified peptides were studied.

The first peptide, KIGDFGMTRDIYETDpYYRKGGK (where pY indicates the

location of phosphotyrosine), was thought to be modified on tyrosine residue 2. For the

ECD experiment the +4 charge state was isolated and subjected to the electron beam.

The resulting ECD spectrum and fragmentation summary can be seen in Figure 3-10.


[M + 4H]4'




K FID LGLMLT R LDo I lYELY LTP pYY1R7K7 K




[M + 4H + e]




300 400 500 600 700 800 900 1000
m /z


Figure 3-10. ECD mass spectrum obtained for KIGDFGMTRDIYETDpYYRKGGK.
The peptide was believed to be modified on tyrosine 2. The resulting ECD
fragmentation allowed for the localization of the modification to the correct
tyrosine residue. Greater than 80% backbone bond cleavage was achieved for
this peptide.

A total of 33 fragment ions were obtained. Cleavage occurred at 19 of 22 possible

backbone bonds, resulting in 86% backbone bond cleavage. The presence of fragment









ions c18 and z6, specifically, indicates that tyrosine residue 2 is indeed modified. There is

no evidence that suggests that any of the remaining residues are modified.

The peptide GnLAGPnLQSpTPLNGARR was the second modified peptide to be

studied using the ECD source. This peptide was predicted to contain a phosphorylated

threonine residue. In this peptide, nL represents norleucine and pT represents

phosphothreonine. Norleucine (D-2-amionhexanoic acid) has the same molecular mass

as leucine (D-2-amino-4-methylpentanoic acid), making differentiation impossible based

upon mass alone. For the ECD experiment, the +3 charge state was isolated by a SWIFT

waveform and fragmented. The resulting ECD mass spectrum and fragmentation

summary can be seen in Figure 3-11.

[M +3H]3


G nLLAGP G PnL QLS pT P LLNLG A R R










JIl iJ 'I .1 il I -
300 400 500 600 700 800 900
m/z

Figure 3-11. ECD mass spectrum obtained for GnLAGPnLQSpTPLNGARR. The
peptide was believed to contain a modified threonine residue. The resulting
ECD fragmentation allowed for the localization of the modification to the
correct residue. Greater than 70% backbone bond cleavage was achieved for
this peptide.

A total of 15 fragment ions were obtained, resulting in the cleavage of 11 out of a

possible 15 backbone bonds. Greater than 70% backbone bond cleavage was achieved.









The presence of fragment ions c9 and z9 indicate that the threonine residue is

phosphorylated. The remaining residues are not modified.

The last modified peptide to be studied was SNKSQKLLRpSPRKPTRKISK.

Serine residue 3 was predicted to be phosphorylated. The ECD mass spectrum obtained

can be seen in Figure 3-12. The resulting fragmentation summary can also be seen in this

figure. A total of 22 fragment ions were obtained during the dissociation event. A total

of 16 backbone bonds were cleaved, resulting in 84% backbone bond cleavage. The

presence of fragment ion zll indicates that serine residue 3 is modified. There is no

evidence to suggest that any of the remaining residues are modified.

[M + 4H]4

S LNLK LSLQ K L L R pS


1T RIKL P T R7 K 1S7 K





[M + 4H + e]3




500 600 700 800 900 1000
m /z


Figure 3-12. ECD mass spectrum for SNKSQKLLRpSPRKPTRKISK. The peptide was
believed to contain a phosphorylated serine residue. The resulting ECD
fragmentation allowed for the localization of the modification to the correct
serine residue. Greater than 80% backbone bond cleavage was achieved for
this peptide.









Conclusions

An electron capture dissociation source was successfully installed on a Finnigan

NewStar Fourier transform mass spectrometer. The use of an El filament as the electron

source was optimized and utilized for the dissociation of various peptide and protein

standards. Studies involving protein digests and modified peptides were also successfully

conducted. The original ECD source was replaced by an indirectly heated dispenser

cathode. The cathode was mounted on the rear axis of the analyzer cell. The electron

energy and irradiation times were optimized for the dissociation of peptides and proteins.

The cathode was successfully utilized to fragment modified peptides. Both the filament

and the cathode allowed for the localization of phosphotyrosine and phosphoserine

modifications. In addition, the use of both CID and ECD were explored. When

combined, the fragment ions obtained from these two dissociation methods provided

greater than 50% backbone bond cleavage for small proteins such as ubiquitin. The main

disadvantage to the use of ECD on this system was the long electron irradiation time.

Upon installation of the dispenser cathode, the irradiation time was lowered by 5 seconds.

However, the use of ECD coupled to liquid chromatography or capillary electrophoresis

is still not practical.














CHAPTER 4
ELECTRON CAPTURE DISSOCIATION STUDIES OF A SERIES OF MARCKS
ANALOGUES

Introduction

Post-translational modifications (PTMs) play vital roles in protein expression.

Eukaryotic proteins undergo various post-translational modifications, with protein

phosphorylation being one of the most widely studied. Protein phosphorylation is a

necessary modification for many protein-protein interactions in cellular recognition,

signal transduction, cell division and cancer. Addition of the phosphate group to a

protein, which results in a net gain in molecular mass of 80 Da, causes conformational

changes in the protein. These changes can alter both the protein's activity and overall

stability. Protein phosphorylation often acts as an on/off switch, controlling many

biochemical functions.173-178 Although the identification of phosphoproteins is possible

with a myriad of biochemical and analytical methods, the subsequent localization of the

site or sites of modification remains quite challenging.

The analysis of phosphoproteins is rather routine but is not always straightforward

from an analytical perspective. The difficulty in analysis can be attributed to five causes.

First, the stoichiometry of phosphorylation is relatively low. Although roughly one-third

of all proteins are phosphorylated, only a small fraction of the actual sample set may be

phosphorylated at any given time. The phosphoproteins may also be heterogeneous.

Several different phosphorylated forms many exist for a phosphoprotein, further

complicating analysis. Many proteins undergo phosphorylation on more than one amino









acid residue. However, all molecules of one protein may or may not be identically

phosphorylated. The phosphoproteins may also be in low abundance within the cells,

making enrichment prior to analysis necessary. In addition, while many major

phosphorylation sites are readily identified by most analytical techniques, the minor sites

of modification may go unidentified. Lastly, the use of phosphatases should be limited,

as these enzymes can dephoshporylate the phosphoproteins during the preparation and

purification steps.24,34,179-182

Although many analytical techniques can be utilized in the analysis of

phosphorylated peptides and proteins, mass spectrometry has become a method of choice.

32P labeling and phosphopeptide mapping studies require large amounts of sample. The

safety hazards associated with these types of experiments can be circumvented by the use

of mass spectrometry. Although the 32P labeling experiments are the most sensitive of

the phosphorylation analysis techniques, the sensitivity of mass spectrometric analyses

(femtomole level) is often more than sufficient for peptide identification. The greatest

advantage to using mass spectrometry over many other techniques is the ability to

localize the sites of modification and obtain full sequence coverage. Phosphopeptide

mapping and Edman degradation do not allow for modification localization. The

sequence coverage associated with these techniques is often low. Full sequence coverage

(100%) for phosphorylated peptides is theoretically possible using a combination of top

down proteomics and Fourier transform mass spectrometry.8'24'38'56'179-182

Electron capture dissociation Fourier transform ion cyclotron resonance mass

spectrometry (ECD-FTICR-MS) has recently emerged as a very powerful method for the

localization of modified amino acids and the sequencing of both modified and









unmodified peptides and proteins.4'5'9'13-15 Several research groups have successfully

applied ECD to the localization of phosphorylated residues in many peptides and

proteins. As stated previously, ECD allows for more extensive fragmentation of the

peptide backbone than collisional dissociation, potentially allowing for greater sequence

coverage. To date, many ECD mass spectrometry experiments have focused on the

identification and localization of modified residues. The ability to identify most modified

residues, with the modification remaining intact during the dissociation process, is the

main advantage of using the ECD technique over the slow heating dissociation methods

such as collision induced dissociation and infrared multiphoton dissociation. The

predicted ion pairs that are formed during both the collision dissociation and electron

capture dissociation processes can be seen in Figure 4-1.

al bl cl a2 b2 c2 a3 b3 c3 a4 b4 c4


H2N-CH- -C- -N- -CH- -C- -N- -CH- -C- -N- -CH- -C- -N- -CH-C--OH
I H I H J H I H I
R1 R2 R 3 R4 _R5
x4 y4 z4 x3 y3 z3 x2 y2 z2 xl yl zl

Figure 4-1. Predicted ion pairs formed during tandem mass spectrometry experiments.
The c/z ion pairs result from the cleavage of the amine bond during ECD
while the b/y ion pairs result from the cleavage of the amide bond during
collisional dissociation. The a/x ion pairs are less common. These ion pairs
are typically only seen for secondary cleavage of the peptide.

For phosphorylated peptides, no loss of phosphoric acid, phosphate or water from

the parent peptide or resulting fragment ions is expected. Because of this, direct

assignment of the phosphorylated (or modified) amino acids can be made. In contrast,

the loss of the modification during collisional dissociation processes makes localization

of the modification very difficult. Data interpretation is typically very time consuming

and tedious for the collisional dissociation experiments.16,118,138,152-157,183,184









As stated in Chapter 1, the MARCKS protein is phosphorylated in the effector

domain region of the protein. This region, also called the phosphorylation site domain

(PSD), contains five serine residues.84'86'88,90 Because each of these residues is capable of

phosphorylation, the localization of the site or sites of phosphorylation may be extremely

difficult. Due to this anticipated difficulty, a series of test peptides (MARCKS

analogues) were synthesized and studied. These peptides were subjected to electron

capture dissociation (ECD) in an attempt to determine the maximum degree of

complexity of multiply phosphorylated polypeptides which could be determined using

ECD. The sequences of the test peptides were modeled after the sequence of the

non-phosphorylated PSD region of the MARCKS protein. The MARCKS analogues

each contain 13 amino acids as opposed to the 25 amino acids present in the original PSD

sequence. The test peptides were synthesized to contain only three serine residues, with a

maximum of two serine residues being phosphorylated at any given time. Because the

synthesis yield of the multiply phosphorylated peptides is traditionally low, the triply

phosphorylated peptides were not produced. The model peptides studied consist of three

singly and three doubly phosphorylated peptides, with a non-phosphorylated peptide used

as a control. The sequences of both the singly and doubly phosphorylated peptides were

identical, with only the number and positions of the phosphate groups being different.

While the fragmentation patterns for each set of test peptides might be expected to be

similar, making complete localization of the modifications somewhat challenging, it was

hoped that fragmentation of these test peptides would provide a series of "fingerprints"

that could then be used to aid in the localization of the sites of modification in more

complex samples. The fragmentation patterns observed for the test peptides could also









provide a series of fragmentation rules that provide a means to determine unambiguously

the sites of modification for many multiply modified polypeptides.

Experimental Methods

Sample Preparation

The seven MARCKS analogues were synthesized at the University of Florida

Protein Core. Phosphorylation was achieved by incorporating phosphoserine residues

into the peptide sequences during the synthesis. The sequences of the seven test peptides

can be seen in Figure 4-2. Purification of the resulting phosphopeptides was achieved by

the use of high performance liquid chromatography (HPLC). The HPLC purification was

performed using a 4.6 mm ID C-18 column, with a flow rate of 1.0 mL/min. Gradient

elution (10-80% B) was utilized (solvent A: 0.1% TFA/H20, solvent B: 0.1%

TFA/ACN). The total gradient time was 32 minutes. MALDI-TOF analysis was used to

confirm the molecular weight of each purified peptide. The MALDI-TOF analyses were

performed on a Voyager DE-PRO MALDI-TOF (Applied Biosystems, Foster City, CA)

operating in reflector mode using delayed extraction. An accelerating voltage of 20,000V

was applied to the ions from each MALDI spot, with an extraction delay time of 200

nsec. The phosphopeptides were analyzed using a matrix composed of ca-cyano-4-

hydroxycinnamic acid. 200 laser shots were acquired per spectrum.

KKSFKLSGFSFKK
KKpSFKLpSGFSFKK KKpSFKLSGFSFKK
KKpSFKLSGFpSFKK KKSFKLpSGFSFKK
KKSFKLpSGFpSFKK KKSFKLSGFpSFKK

Figure 4-2. The sequences of MARCKS analogues 1 through 7. Peptide 1 is non-
phosphorylated (top), peptides 2-4 are doubly phosphorylated (left) and
peptides 5-7 are singly phosphorylated (right). The modified serine residues
are highlighted in red.









Basic Fourier Transform Mass Spectrometry

The MARCKS analogues were analyzed on a 4.7T Bruker 47e FTICR mass

spectrometer (Bruker Daltonics, Billerica, MA). Ionization was achieved by the use of an

electrospray ionization source retrofitted with a heated metal capillary 85,186 (Analytica,

Branford, CT). An instrumental schematic can be seen in Figure 4-3.



Ion transfer optics
I I trs 4.7T superconducting magne





ESI


Turbomolecular pumps ICR cell



Figure 4-3. Schematic of the Bruker 47e FTICR mass spectrometer equipped with an
Analytica electrospray ionization source and an external hexapole for ion
accumulation.

The phosphopeptides were electrosprayed in positive ion mode at a flow rate of 333

nL/min using a ground fused silica microspray tip. Final solution concentrations were

5-10 [iM in 1:1 acetonitrile:water. The addition of 1% (by volume) acetic acid promoted

the formation of the positively charged ions. The heated metal capillary was held at a

temperature of 120 C and the electrospray voltage was between 1500V and 2500V for

all experiments. The positively charged ions were accumulated for 0.7 seconds in an

external hexapole prior to transfer to the analyzer cell. All spectra were acquired in

positive ion mode, with 32 single scan acquisitions averaged per spectrum. Unless noted









otherwise, 256K data points were collected and processed, with broadband detection

being used in all experiments.

Electron Capture Dissociation Fourier Transform Mass Spectrometry

All electron capture dissociation experiments were performed on a home-built 9.4T

ESI-Q-FTICR mass spectrometer at the National High Magnetic Field Laboratory

(NHMFL). The instrument schematic can be seen in Figure 4-4. Sample introduction

was achieved using a Nanomate robot (Advion Biosciences, Ithaca, NY) operating at a

flow rate of 200 nL/min. Final solution concentrations for the electrospray ionization

process were 5-10 [M in 1:1 acetonitrile:water with the addition of 0.1% formic acid.

Approximately 40 [L of sample was loaded into each well of the Nanomate well plate.

The total sample consumption was less than 15 gL. The phosphopeptide ions were

accumulated for 1 second in the middle octopole.

Transfer octopoloe
Front octopole L IRIPD
_Iiddle "
Sl Dispenser
SQuadrupole octopole ICR cell cathode
SI ell cathode


I "/ i \ i
SI _______ -
-- __- I___1--I--I1" ."

-10-3 Torr

Turbomolecular pumps

Figure 4-4. Home-built 9.4T FTICR mass spectrometer at the NHMFL. This system is
equipped with a dispenser cathode for ECD experiments (on-axis) and a CO2
laser for IRMPD experiments (off-axis). Figure adapted from reference 160.

The desired charge state for fragmentation was isolated by an external

mass-selective quadrupole mass filter. No SWIFT (stored waveform inverse Fourier









transform) isolation was utilized. The ECD experiments were performed using a 10mm

BaO coated dispenser cathode (HeatWave, Watsonville, CA) mounted on the rear axis of

the analyzer cell. The ECD source operated at a potential of-5V during the ECD events.

The electron irradiation time was 20ms, with an electron energy of 5 eV. Unless stated

otherwise, 512K data points were collected and processed. A total of 100 single scan

acquisitions were averaged for each tandem mass spectrum.

Data Analysis

Data analysis was achieved using the MIDAS (Modular ICR Data Acquisition

System) data analysis software developed at the NHMFL.114 Each spectrum was baseline

corrected and zero filled twice. Hanning apodization was utilized during the data

processing step.168 The THRASH program (Thorough High Resolution Analysis of

Spectra by Horn) was used to generate the experimental peak lists obtained for the ECD

experiments.169 The monoisotopic masses obtained for each fragment ion were imported

into the ProSight PTM program (Kelleher group, UIUC) for generation of the

fragmentation summaries for each phosphopeptide.170'171 A mass tolerance of 0.1 Da was

used when identifying the fragment ions. The ProSight program was used to positively

identify the c/z and b/y ion pairs formed from the fragmentation of the MARCKS

analogues. The a-type ions were identified from the Protein Prospector database

(UCSF).187

Results and Discussion

Prior to performing the electron capture dissociation experiments at the NHMFL,

the seven MARCKS analogues were analyzed as further confirmation of the

monoisotopic mass of each peptide (Table 4-1).










Table 4-1. Predicted monoisotopic masses for the seven MARCKS analogues.
Peptide Monoisotopic Mass (Da)
KKSFKLSGFSFKK 1530.8922
KKpSFKLpSGFSFKK 1690.8248
KKpSFKLSGFpSFKK 1690.8248
KKSFKLpSGFpSFKK 1690.8248
KKpSFKLSGFSFKK 1610.8585
KKSFKLpSGFSFKK 1610.8585
KKSFKLSGFpSFKK 1610.8585

It should be noted that MARCKS analogues 2-4 have the same monoisotopic mass,

as do MARCKS analogues 5-7. The spectrum obtained for the mass spectrometric

analysis of the non-phosphorylated peptide is shown in Figure 4-5.

[M + 3H]3+








[M + 2H]2+





400 600 800
m /z

Figure 4-5. FTICR mass spectrum obtained for the non-phosphorylated MARCKS
analogue. The +2 and +3 charge states were present in the positive mode
analysis of this peptide. The remaining peaks are most likely due to the
chemicals and buffers used during the synthesis.

Both the +2 and +3 charge states are present in the spectrum. Figure 4-6 shows the

spectra obtained for the analysis of MARCKS analogues 2-4. These three peptides

contain two sites of phosphorylation, making differentiation between these peptides

impossible based upon molecular mass alone. In each case, the +2 charge state was also

observed. The +3 charge state was only observed for peptides 2 and 3.











A) M 2] B) ,[M + 2H]M C 2H)


[M + 3H ] s+






Figure 4-6. FTICR mass spectra obtained for the doubly phosphorylated MARCKS
analogues. Differentiation between these three peptides cannot be made upon
mass analysis alone. A) MARCKS analogue 2. B) MARCKS analogue 3.
C) MARCKS analogue 4.

A similar scenario was seen for the singly phosphorylated MARCKS analogues 5-

7. The resulting mass spectra can be seen in Figure 4-7. Once again, differentiation

between the singly phosphorylated peptides cannot be made based upon mass alone. To

differentiate between the two sets of phosphopeptides (3 singly phosphorylated, 3 doubly

phosphorylated), a series of electron capture dissociation experiments were carried out.



A ) [M + 2H] B) [M + 3H] [M+ [M2 3H
[M + 2H2



400 600 800 0 600 800 800 4 600 8 00


Figure 4-7. FTICR mass spectra obtained for the mass analysis of the singly
phosphorylated MARCKS analogues. Each peptide has the same mass,
making differentiation difficult. A) MARCKS analogue 5. B) MARCKS
analogue 6. C) MARCKS analogue 7.

Fragmentation of the Non-phosphorylated Peptide

For the electron capture dissociation studies of the non-phosphorylated peptide

(MARCKS analogue 1), the [M + 3H]3+ charge state was isolated in the external

quadrupole mass filter for 1 second prior to fragmentation. Upon transfer to the analyzer










cell, the desired ions were subjected to electron irradiation for 20 milliseconds. The

capture of an electron by the +3 charge state is indicated by the presence of m/z 766 ([M

+ 3H + e-]2+) in the resulting spectrum. The presence of this ion signifies that electron

capture has occurred and that the fragment ions are a direct result of the electron capture

dissociation process. As can be seen in Figure 4-8, all backbone bonds were cleaved for

the non-phosphorylated peptide, giving complete sequence coverage.

[M 3H]3+
K LK Ls LFKL K IL SILGLJFIS ~FKs FK K




[M + 3H+e] +



[M + 4H]4 c a

C

+Z

C + a. a,

J A17l .... 10; I8 12+
400 600 800 1000 1200 1400 1600
m/z


Figure 4-8. ECD mass spectrum obtained for the dissociation of MARCKS analogue 1.
The fragmentation summary contains a-ions (blue), y-ions (green) and c/z ion
pairs (red). All backbone bonds were cleaved, giving 100% sequence
coverage.

A total of 9 c-type ion and 10 z-type ions were observed. A series of 7 a-type ions

were also observed. In addition to the typical ECD fragment ions (a, c, z-type ions), 5 y-

type ions were also obtained. The resulting fragmentation summary, showing all

observed fragment ions, can be seen in the inset of Figure 4.8.










Fragmentation of Singly Phosphorylated Polypeptides

For each of the singly phosphorylated peptides, the [M + 3H]3+ charge state was

isolated in the external quadrupole mass filter prior to fragmentation. MARCKS

analogue 5 contains three serine residues. Serine residue 1 is phosphorylated, while the

remaining serine residues contain no modification. Figure 4-9 shows the ECD mass

spectrum and resulting fragmentation summary obtained for peptide 5. Cleavage

occurred at all backbone bonds, resulting in the formation of 10 c-type and 10 z-type

ions. In addition to the traditional ECD fragment ions, 9 a-type ions and 6 y-type ions

were formed during the electron capture dissociation process.



K LK LpS]L FL LK L L G]LFnS FLLK K





[M + 3H + e]2
[M + 3H]3'
a H


I zz





400 600 800 1000 1200 1400 1600
m /z


Figure 4-9. ECD mass spectrum generated for MARCKS analogue 5. This peptide is
phosphorylated at serine residue 1. 100% sequence coverage was obtained for
this peptide. The fragment ions are denoted by the same colors as in Figure 4-
8.

The presence of fragment ions on both the N-terminal (zii) and C-terminal (a3, c3)

sides of the modified residue serinee 1) indicate the presence of a phosphate group. There

is no indication that either serine 2 or serine 3 is modified.










The ECD mass spectrum for MARCKS analogue 6 can be seen in Figure 4-10. In

this case, all backbone bonds were cleaved, allowing for localization of the modification

to serine 2. As seen in Figure 4-10, the presence of specific fragment ions (z7, c7, a7)

allow for the unambiguous localization of the lone modification to serine 2. There is no

evidence that either serine 1 or serine 3 is modified.

[M + 3H]3

K K L pLGILF S FM K K






[M + 3H + e ]







400 600 800 1000 1200 1400 1600
m/z

Figure 4-10. ECD mass spectrum generated for the fragmentation of MARCKS analogue
6. This peptide is phosphorylated at serine residue 2. Complete sequence
coverage was obtained. The fragment ions in the summary are denoted by the
same colors as in Figure 4-8.

Figure 4-11 shows the ECD mass spectrum and fragmentation summary for

MARCKS analogue 7, which contains a modification on serine 3. A total of 34 fragment

ions were observed. The presence of fragment ions z4, a10 and clo allow for localization

of the modification to this particular residue. There is no indication that series 1 or 2 are

modified. The fragmentation summaries obtained for each of the singly modified

polypeptides allowed for the localization of the modification to the predicted residue.









Fragmentation of Doubly Phosphorylated Polypeptides

Figure 4-12 shows the ECD mass spectrum for MARCKS analogue 2. This peptide

contains sites of modification on series 1 and 2. There is no modification on serine 3.

Cleavage occurred at all backbone bonds, allowing for complete sequencing of this

phosphopeptide. A total of 34 fragment ions were formed. The resulting fragmentation

summary can also be seen in Figure 4-12.


'K LK LL SLL L iL GL LL P~ S1 F] K 7K

[M + 3H]3
S [M + 3H + e]2'

z+ z1 Y1 a,

z'



I,, + z\11


400 600 800 1000 1200 1400 1600
m/z

Figure 4-11. ECD mass spectrum obtained for the dissociation of MARCKS analogue 7.
Serine residue 3 is phosphorylated. Complete sequence coverage was
obtained for the dissociation of this peptide. The fragment ions are denoted
by the same color scheme as in Figure 4-8.

From this summary, localization of the sites of modification is possible. The

fragment ions on the N-terminal (z11) and C-terminal (c3) sides of serine 1 indicate the

presence of a phosphate group. The presence of fragment ions z7 and c7 indicate the

presence of a modification on serine 2. The fragment ions surrounding serine 3 show an

unmodified residue at this position.










The ECD mass spectrum and fragmentation summary for MARCKS analogue 3

can be seen in Figure 4-13. As with MARCKS analogue 2, cleavage was seen for all

backbone bonds. The fragmentation summary for peptide 3 shows a series of 10 c-type

ions and 9 z-type ions, along with 7 a-type ions. Seven y-type ions were also observed.

[M + 3H]'+

K LK LPS LFSLK LL|pS LG LY LsL LFYK IK







[M + 3H + e]
C l
I c, +






400 600 800 1000 1200 1400 1600





denoted by the same color scheme as in Figure 4-8.

These fragment ions allow for the localization of the phosphate groups to series 1

and 3, as expected. Specifically, fragments ions z1 and c3 indicate the presence of a

modification on serine I while fragment ions z4 and co indicate the presence of a

modification on serine 3. The fragment ions obtained do not indicate any sort of

modification on serine 2.

Figure 4-14 shows the spectrum from MARCKS analogue 4. This peptide is

phosphorylated on series 2 and 3. The presence of fragment ions on both the N-terminal

and C-terminal sides of both series 2 and 3 allow for the localization of a modification
and C-terminal sides of both series 2 and 3 allow for the localization of a modification










on each of these residues. The fragment ions obtained do not show any modification on

serine 1.


[M + 3H]3'
K K LpS LFLKI LL LiGILF1 PpS LlFlK IK






[M + 3H + e ]



c' +z



400 600 800 1000 1200 1400 1600
m/z


Figure 4-13. ECD mass spectrum obtained for the dissociation of MARCKS analogue 3.
Serines 1 and 3 are phosphorylated. Complete sequence coverage was
obtained for this peptide. The fragment ions are denoted by the same color
scheme as in Figure 4-8.

Differentiation of Polypeptides with Identical Mass and Number of Modifications

As seen in Figure 4-2, MARCKS analogues 2, 3 and 4 have the same mass and

number of modifications. An identical scenario can be seen for MARCKS analogues 5, 6

and 7. Because of this, definitive assignment of the sites of modification may not always

be straightforward. As stated earlier, it is anticipated that it will be difficult to distinguish

between peptides that contain the same number of phosphate groups and have the same

molecular mass. Because of this, a study was undertaken to determine if it is indeed

possible to differentiate between the ECD data sets of each peptide.










[M + 3H]

K K LSLF]L iLLp SLGLTF ]pS FGLK 7K







[M + 3H+ e]
a *
+ I2






400 600 800 1000 1200 1400 1600
m/z


Figure 4-14. ECD mass spectrum obtained for the dissociation of MARCKS analogue 4.
Serine residues 2 and 3 are modified. 100% sequence coverage was obtained
for this peptide. The fragment ions are denoted in the fragmentation summary
using the same color scheme as in Figure 4-8.

The experimental peak lists obtained for each of the doubly phosphorylated

peptides were compared to those predicted for each of the doubly modified peptides in

turn to determine how many of the experimental fragment ions are identical for each

peptide.

Based upon the theoretically obtained peak lists, several fragment ions for each of

the doubly modified peptides should be identical, with the remaining ions being different

for each of the respective peptides. The fragment ions that are unique to each peptide

should only be in the experimentally generated peak lists for the "correct" peptide. As an

example, fragment ions zll and c3 should be identical for peptides 2 and 3 while fragment

ions C7 and z5 should be different. The fragment ions that change from peptide to peptide

should provide a means to unambiguously differentiate between each of the similar

peptides. An analogous scenario was seen for the singly phosphorylated peptides as well.