Citation
Mass Spectrometric Analysis of Two Phosphorylation-Based Signal Transduction Systems: Site-Specific Effects of the Circadian Clock on Limulus Myosin III Phosphorylation, and Binding Selectivity of the Arabidopsis Family of 14-3-3 Isoforms

Material Information

Title:
Mass Spectrometric Analysis of Two Phosphorylation-Based Signal Transduction Systems: Site-Specific Effects of the Circadian Clock on Limulus Myosin III Phosphorylation, and Binding Selectivity of the Arabidopsis Family of 14-3-3 Isoforms
Creator:
CARDASIS, HELENE L. ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Actins ( jstor )
Ionization ( jstor )
Ions ( jstor )
Isotopes ( jstor )
Mass spectrometers ( jstor )
Mass spectroscopy ( jstor )
Phosphorylation ( jstor )
Protein isoforms ( jstor )
Quadrupoles ( jstor )
Signals ( jstor )

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Helene L. Cardasis. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2007
Resource Identifier:
649810173 ( OCLC )

Downloads

This item is only available as the following downloads:


Full Text

PAGE 1

MASS SPECTROMETRIC ANALYSIS OF TWO PHOSPHORYLATION-BASED SIGNAL TRANSDUCTION SYSTEMS: SITE SPECIFIC EFFECTS OF THE CIRCADIAN CLOCK ON Limulus MYOSIN III PHOSPHORYLATION, AND BINDING SELECTIVITY OF THE Arabidopsis FAMILY OF 14-3-3 ISOFORMS By HELENE L. CARDASIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

PAGE 2

Copyright 2006 by Helene L. Cardasis

PAGE 3

For Mom and Dad. For Yaya.

PAGE 4

iv ACKNOWLEDGMENTS I will begin with my beginnings . . . Above a ll, I want to thank my parents. They are my strength, my support, my motivation to improve. They have equipped me with the things I needed most to make it through th is experience: the abil ity to learn from my mistakes, the humility to laugh at myself, and th e persistence to “try, try again.” Even today, as I near the completion of this challe nge and can see the reward within my reach, I can honestly state without hesitation that I am prouder of the tit le "Ted and Monica's daughter" than any title or honor I will ever earn. I also thank my brother, Ted, who has gr ounded me when I needed it most. He reminds me that I am not the smart one in the family, encourages me to be a "big brave dog" (Chuckie, Rugrats; 1997), and makes me laugh like no one else can. We are one and the same, and completely different. E ither way, he is truly my best friend. My grandmother, Yaya, was and is my smiling face through every situation, my definition of strength, and my inspiration. She saw the silver lin ing in every cloud, and found every day “beautiful, beautiful.” I cons ider myself extremely lucky to have known her so well and to have learned so much from her. She is ever present in my heart and mind, and her memory will always bring perspective to my life. I have also been lucky enough to have some great friends who have always supported me and kept me laughing. I want to first thank my undergrad crowd and second family, Steve, Eileen, Fro, and Jess, fo r always pushing me to prove that “yes, I am a warrior.” I can’t imagin e who I’d be today without th em. To Tiffany, my thanks

PAGE 5

v for making grad school fun, fo r understanding, and for keeping me constantly laughing. To Anthony, who grounded me, supported me, and was always there when I needed him, go my deepest thanks. To Sue, my thanks for her never-ending encouragement and enthusiasm. And finally, to Travis and Jos h, who exemplified my reason for being here (what is fire?), my thanks for making me to thi nk, reminding me to relax, and training me show up on time. Without further delay, I want to extend my deepest thanks to my very own “team of specialists” that got me through the last five years. Great thanks go to Dr. Dave Powell for taking me into the “orphanage.” Being our (the orphans) adviso r was not in his job description, but he took on the job with ent husiasm and care. Thanks also go to my ‘mass spec mom,’ Dr. Lidia Matveeva, who taught me to think, and then think again. She instilled in me the fundamentals I needed to move forward in this program, and in life, and for that I am foreve r grateful. She is deeply missed. I also owe immeasurable thanks to Dr. Jodie Johnson, whose enthusia stically frank conversations concerning mass spec (or anything) were alwa ys refreshing, and extremely informative and enlightening experiences for me. I also offer my deepest appreciation to Dr. John Eyler, my advisor, who always made himself available to discuss anything at all, from statistics to ion optics to experimental planning. His support and guidance during my time in his group are much appreciated. I owe great thanks to Dr. Stan Stevens, whose clear understanding of theory and applications alike set a high standard for Eyler group graduates who follow him. I truly appreciate the opportunity he provided me through my employment at the Protein Core, as well as his guidance and advisement during my extremely beneficial year in his

PAGE 6

vi lab. My success and growth at the Protein Co re are also attributed to many invaluable conversations with Scott McClung. I tha nk him for challenging me, for making me defend my ideas, and for advocating my educat ion whenever possible. I would also like to thank Dr. Keith Zeintek for his help in editing this dissertation, and for fervently discussing FTICR-MS whenever prompted. He will be a great teacher one day. I would also like to thank the remaini ng members of my committee, Dr. Richard Yost, Dr. Tom Lyons, and Dr. Rob Ferl, for th eir scientific advisement and support. From Dr. Ferl’s lab, I extend great thanks to Dr. Paul Sehnke for his advise and helpful discussions, Dr. Mike Manak fo r his help with protein pu rification, and Matt Reyes for his intriguing conversations concerning 14-3-3 biology. Dr. Barbara-Anne Battelle has been a true mentor to me from the time of my undergraduate internship and throughout graduate school. I deeply admire her for her dedication, her love for her field and for scie nce in general, and the enthusiasm for her work that she demonstrates everyday. I cons ider myself extremely lucky to have worked with her and hope to always be able to work with her on some level. I simply cannot thank her enough for her support, inspiration, and guidance. From Dr. Battelle’s lab, I would also like to thank Karen Kempler, who has been a great help to me from my first days in the lab as an undergraduate to now. Finally, I would like to thank the teachers who made such a huge difference early on and encouraged me to follow my interest s and curiosities. To Mr. Campisi, Mr. Rinella, Mr. Bollerud, Mr. Cheverton, Dr. Lang, Dr. Strange, and Dr. Evansthank you for teaching.

PAGE 7

vii TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 Protein Phosphorylation................................................................................................2 Analytical Challenges in the Study of Protein Phosphorylation...........................3 Protein Phosphorylation Analysis Techniques......................................................4 Mass Spectrometry in Protein Phosphorylation Analysis............................................8 Ionization of Phosphorylated Peptides and Proteins.............................................8 Electrospray ionization...................................................................................9 Alternative ionization methods: MALDI, ICP.............................................12 Mass Spectrometric Methods of De tecting Protein Phosphorylation..................13 Phosphopeptide/ Protein Enri chment and Separation.................................................18 Immobilized Metal Ion Af finity Chromatography..............................................18 Titanium Dioxide Enrichment.............................................................................21 -elimination/ Mi chael addition..........................................................................22 Cation/Anion Exchange for Phosphopeptide Fractionation................................23 Hydrophobicity-based Adsorption Techniques...................................................23 Protein and PTM Quantitation by Mass Spectrometry...............................................24 Relative Quantitation...........................................................................................24 Absolute Quantitation..........................................................................................26 Summary.....................................................................................................................27 2 INSTRUMENTATION..............................................................................................29 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.............................29 Quadrupole Ion Trap Mass Spectrometry...................................................................35 Hyrbid Quadrupole-Time of Flight (qTOF) Mass Spectrometry...............................40

PAGE 8

viii 3 SITE SPECFIC EFFECTS OF THE CIRCADIAN CLOCK ON LIMULUS MYOSIN III PHOSPHORYLATION........................................................................45 Methods......................................................................................................................50 Animals and Standard Reagents..........................................................................50 Lateral Optic Nerve Sections, La teral Eye Extraction, and LpMyoIII Purification.......................................................................................................51 LpMyoIII Digestion.............................................................................................51 Methyl Esterification – Differential Labeling.....................................................52 LC-MS/MS analysis............................................................................................53 Differential-Labeling Da ta Interpretation............................................................54 Label-Free Data Interpretation............................................................................58 Results........................................................................................................................ .58 Verification and Identification of in vivo LpMyoIII Phosphorylation Sites.......58 Validation of Quantitation Methods....................................................................63 Changes in Levels of Phosphorylation in Response to Circadian Efferent Input (Clock Input)..........................................................................................65 Discussion...................................................................................................................69 Method Development for Measuri ng Changes in Phosphorylation....................70 Identification of in vivo Phosphorylation Sites in Endogenous LpMyoIII..........73 Measurement of Site Specific in vivo Changes in Phosphorylation at PKA Sites in Response to Circadian Cl ock Input – Assigning Function to Phosphorylation?..............................................................................................74 Significance of Phosphorylation at S-796 and S-846..........................................75 Possible Functional Consequences of Phosphorylation at S-796 and S-846 for the function of Limulus photoreceptors...........................................................76 Future Directions........................................................................................................78 Results of Preliminary Assays of Light -Regulated LpMyoIII Phosphorylation........79 Summary.....................................................................................................................81 4 MASS SPECTROMETRIC EVALUATI ON OF 14-3-3 ISOFORM BINDING SELECTIVITY...........................................................................................................82 Introduction to the 14-3-3 Family of Proteins............................................................82 Methods......................................................................................................................89 Protein Expression and Purification.............................................................89 Phosphopeptide Synthesis...................................................................................90 Buffer Optimization for ESI-MS Coupl ed Microaffinity Chromatography........90 14-3-3 Microaffinity Capture Chromatography..................................................91 Mass Spectrometric Instrumentati on and Operational Parameters.....................92 Results........................................................................................................................ .93 Optimization of Affinity Capture Experiment for Mass Spectrometric Coupling...........................................................................................................93 Effects of Loop 8 Point Mutati on on Phosphopeptide Binding..........................96 Analysis of 14-3-3 Sub-Group Speci fic Target Phosphopeptide Sequence Preferences.......................................................................................................99 Discussion.................................................................................................................105

PAGE 9

ix Future Directions......................................................................................................107 Summary...................................................................................................................110 5 CONCLUDING REMARKS....................................................................................111 Class III Myosins: From Horseshoe Crabs to Humans....................................113 The 14-3-3 Protein Family: Fr om Common Weeds to Humans......................115 LIST OF REFERENCES.................................................................................................118 BIOGRAPHICAL SKETCH...........................................................................................130

PAGE 10

x LIST OF TABLES Table page 3-1 Representative determination of to tal peak areas for two phosphopeptides for both the (+)clock and (-)clock cell state, and subsequent calculation of raw (+)clock/ (-)clock ratios............................................................................................56 3-2 Representative determination of total p eak areas for four control peptides in the (+)clock and (-)clock cell states, and the subsequent calculation of control (+)clock/ (-)clock ratios............................................................................................57 3-3 Error analysis applie d to control peptide (+)c lock/ (-)clock ratios..........................57 3-4 Normalization of phosphopeptide raw (+)clock/ (-)clock ratios and error propagation from control peptide e rror analysis to final values...............................57 3-5 Raw (+)clock/(-)clock ratios, aver age normalization ratios, relative 95% confidence intervals, and normalized (+)clock/(-)clock values for S-841 and S846 from each experimental set...............................................................................66 3-6 Raw (+)clock/(-)clock ratios, aver age normalization ratios, relative 95% confidence intervals, and normalized (+)clock/(-)clock values for S-796, S-841, and S-846 from each set...........................................................................................69 4-1 Average “before binding” phosphopeptide/ WT ratios, average “after binding” phosphopeptide/WT ratios, their associat ed standard deviations, and final “binding ratios” with prop agated error analysis.....................................................104

PAGE 11

xi LIST OF FIGURES Figure page 1-1 Formation of “mini” Taylor cone from ESI-produced charged droplets. ................9 2-1 Configuration of trapping, excitatio n, and detection plates in ICR cell...................30 2-2 Ion cyclotron motion................................................................................................31 2-3 Configuration of electrode s in 3D quadrupole ion trap. .........................................36 2-4 The Mathieu stability diagram describing ion stability in a quadrupole ion trap....37 2-5 Schematic of the Applied Biosys tems QSTAR XL hybrid qTOF MS....................40 3-1: Schematic representation of the signaling cascade resp onsible for circadian clockdriven changes in visual function in Limulus ...........................................................46 3-2 Immunostaining of myosin III and ac tin in cross sections of lateral eye ommatidia.................................................................................................................50 3-3 Schematic of both label-free and differential labeling methods...............................53 3-4 Representative calculation of (+) clock to (-) clock ratio via differential labeling method......................................................................................................................55 3-5 MS/MS verification of four sites in LpMyoIII phosphorylated in vivo...................60 3-6 Methyl esterificati on reaction efficiency..................................................................63 3-7 Schematic of standard sample prepar ation scheme (upper panel) and validation of label free relative quantit ation method (lower panel). In schematic, P = phosphopeptide; A,B,C = BSA tryptic control peptides..........................................64 3-8 Normalized (+) clock to (-) clock ratios calculated from three independent animal sets via differential labeling.........................................................................66 3-9 Comparison of average results from differential labeling and label-free experiments..............................................................................................................67 3-10 Final (+) clock to (-) clock ratios of three sites phosphoryla tion sites, indicating the degree to which phosphorylation cha nges in response to clock input...............69

PAGE 12

xii 3-11 Justification for integration of first two isotope peaks as a more accurate means of ratio determination...............................................................................................72 3-12 Ommatidia cross sections from a late ral eye which had received optic nerve input (control) and one to which the optic nerve had been severed (ONS).............77 3-13 Preliminary (+)light to (-)light ratios determined by label free relative quantitation for each of three phosphorylation sites identified in th e lateral eye. ..80 4-1 Structural diagram of a 14-3-3 dimer.......................................................................84 4-2 Effect of MES on electrospray io nization efficiency of phosphopeptides. ............94 4-3 Effects of ammonium bicarbonate c oncentration on electrospray ionization efficiency of synthetic phosphopeptides..................................................................95 4-4 Determination of number of salt washes necessary post-phosphopeptide incubation to eliminate non-specific binding of phosphopeptides to the Ni2+ charged resin............................................................................................................96 4-5 Representative direct infusion ESI-F TICR-MS spectrum of the “before binding” phosphopeptide mixture...........................................................................................97 4-6 ‘After-binding’ phosphopeptide profile s of a non-epsilon (A ) and epsilon (C) isoform, along with their G/N213-mutate d counterparts in panels B and D, respectively...............................................................................................................98 4-7 Schematic diagram of experimental work-flow for quantitation of relative binding affinities of phosphopeptides to specific 14-3-3 isoforms........................100 4-8 Plot of approximate phosphopeptid e concentration vs. phosphopeptide/WT signal intensity ratio...............................................................................................101 4-9 Plot of approximate phosphopeptide concentration Vs. each phosphopeptide/ WT* signal intensity ratio......................................................................................102 4-10 Determination of LLOQ.........................................................................................102 4-11 Relative measure of each phosphopeptide’ s binding affinity to each isoform, normalized against the NR WT peptide.................................................................104 4-12 Demonstration of phosphopeptide separa tion by reversed phase capillary HPLC and subsequent ion trap MS detection....................................................................109

PAGE 13

xiii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MASS SPECTROMETRIC ANALYSIS OF TWO PHOSPHORYLATION-BASED SIGNAL TRANSDUCTION SYSTEMS: SITE SPECIFIC EFFECTS OF THE CIRCADIAN CLOCK ON Limulus MYOSIN III PHOSPHORYLATION, AND BINDING SELECTIVITY OF THE Arabidopsis FAMILY OF 14-3-3 ISOFORMS By Helene L. Cardasis August 2006 Chair: John R. Eyler Major Department: Chemistry The mass spectrometric evaluation of two phosphorylation-based signal transduction systems is presented here. Both analyses involved the relative measurement of change, either in levels of phosphorylati on in response to a stimulus or in binding affinities as a result of structural va riation. In the first system, that of Limulus polyphemus Myosin III (a circadian clock-regulated phosphoprotein in the lateral eyes of these animals), a quadrupole ion trap ma ss spectrometer with a home built in-line capillary HPLC system enabled the identific ation and verification of four sites of in vivo phosphorylation. Subsequently, a hybrid qua drupole-time of flight mass spectrometer with an in-line integrated cap illary HPLC system was utilized to measure changes in the levels of phosphorylation at each of these sites in response to circadian clock input. Two of these sites demonstrated a reproducibl e increase in levels of phosphorylation in

PAGE 14

xiv response to this neural stimulus. Localiza tion of these clock-regulated phosphorylation sites to a region of myosin known to participate in actin binding lends additional support to the hypothesis for a novel mechan ism of acto-myosin interaction. In the second system, that of the Arabidopsis 14-3-3 family of phosphoprotein/ peptide-binding proteins, an FTICR mass spectrometer with a direct infusion ESI source was utilized to profile differences in the binding of eight synthetic octamer phosphopeptides to different 14-3-3 isoforms. Profiling of two isoforms representing the two major sub-groups of the family of 14-3-3s and their respective single point mutants revealed preliminary evidence for a possible role of the mutated residue in substrate selectivity. This experiment also demons trated the need for a more comprehensive characterization of sub-group binding selec tivity prior to mutation. A method for the relative quantitation of phosphopeptide binding affinities was developed to characterize the binding preferences of any 14-3-3 isoform and/or 143-3 mutant. Two isoforms representative of the tw o major sub-groups of the Arabidopsis 14-3-3 family were selected for analysis by this relative qua ntitation method. Repr oducible selectivity among the phosphopeptide sequences was observed in an isoform-independent manner.

PAGE 15

1 CHAPTER 1 INTRODUCTION The last century has witnessed an ever-ac celerating evolution of mass spectrometry as an analytical tool. From its inception, ma ss spectrometry has remained at the forefront of technology. It has enabled us to challenge old theories a nd create new ones, constantly testing the minds of those who work ed to develop and apply it. Its notable first contribution to science, th e discovery and charac terization of stable element isotopes, brought keen attention to the field. As the scie ntific community took notice, improvements to instrument desi gn and performance not only increased the accuracy and precision of measurements for the academic physicists who had developed the technique, but simultaneously expanded its realm of application. Biologists and geologists soon recognized the value of usi ng natural isotopes to follow and understand phenomena in their own fields, and mass spect rometry slowly emerged as an important tool in these labs. Oil companies iden tified mass spectrometry’s unique ability to differentiate hydrocarbon isomers far faster and with less sample requirement than traditional methods, and mass spectrometry was soon thrust into industrial and commercial settings as well. World War II’s demand for immediate advancements in technology pushed the field forward even fu rther. Using preparative scale mass spectrometry, Uranium-235 was identified as th e sole isotope of ur anium that undergoes high energy fission. Shortly thereafter, new high precision/high sensitivity instruments were used to monitor the process of U-235 collection at the Oak Ridge gaseous diffusion plant as construction of the atomic bomb went underway.

PAGE 16

2 Mass spectrometry has found a pplication in everything from medicine to quantum mechanics. With the various types of mass analyzers, ionization sources, and front end separation techniques available commercially to researchers, systems can be assembled to specifically serve a particular t ype of need. The applications that will be the focus of this dissertation concern the qualitative and se mi-quantitative analysis of two unique phosphoprotein systems and their role in phos phorylation-based signa l transduction in those systems. The significance of each of th ese systems will be individually discussed in detail in their respective chapters. These applications represent an important role mass spectrometry is currently playing in the elucidation of complex in vivo biochemical signal transduction schemes, a topic important to those studying bot h the physiology and pathology of different biological systems (Grayson et al. , 2002). Protein Phosphorylation The reversible phosphorylation of a protei n can have a wide array of downstream effects within a cell, including, but not limite d to, regulation of ce llular proliferation, differentiation, metabolism, sub-cellula r localization, and apoptosis (Peters et al. , 2004; Moore and Sefton, 2004). By eith er activating or inactivating a particular action of the protein substrate, protein phosphor ylation acts as a biological “switch” that turns on or off that action. This can be achieved by either a change in conformation that causes local or allosteric changes in binding affinity to a secondary substrate, a change in the local chemical environment (i.e. pH, negative charge ) which in turn alters its binding affinity to a secondary substrate, or by sterically bloc king local binding of a secondary substrate. The reversible nature of this covalent post translational m odification (PTM) enables a cell to actively adapt to environmental and/or physiological stimuli and transmit signals throughout the cell that reflect the changing ce llular requirements. With this in mind, it

PAGE 17

3 becomes apparent that the successful transfer of these signals is cr itical to proper cell function, and likewise the failure of any one protein in a signal transduction cascade to accept or transmit the signal can lead to a myriad of cellular pathologies. Phosphorylation and dephosphorylation of a protein are catalyzed by protein kinases and phosphatases, respectively. An estimated 1000 kinases and 500 phosphatases are encoded by the human genome (Peters et al. , 2004), lending perspective as to the complexity of cellular control of wh en, where, and why a protein becomes phosphorylated or dephosphorylated. Even w ith this “control by diversity,” it is estimated that approximately on e third of all proteins are re gulated by particular kinases and phosphatases at some point in their life cycle, and therefore play some type of role in phosphorylation-based signa l transduction (Peters et al. , 2004). Analytical Challenges in the St udy of Protein Phosphorylation Despite nature’s universal use of phosphoryl ation as an important regulatory post translational modification, the an alysis of this dynamic event is far from simple. While effects of protein phosphoryla tion can be widespread and lo ng-lived, the event itself can be extremely transient and highly localized to only a small portion of a given protein population, resulting in low overall stoichiometry of the modified protein. In addition, phosphorylation at different site s on a protein may elicit differe nt effects within the cell, making it crucial to be able to temporally distinguish heterologous phosphoprotein forms when studying the biological effect s of each phosphorylation event. Researchers in the field of phosphoproteomics seek to answer four basic questions: 1) Which proteins within a system become phosphorylated? 2) Where , or at what specific sites, does a particular pr otein become phosphorylated? 3) When , or in response to what environmental and/or physiological st imuli, does each site on that protein become

PAGE 18

4 phosphorylated? 4) How much , or to what degree, is each site phosphorylated within the protein population at any particular time? Protein Phosphorylation Analysis Techniques Radiolabelling. A number of techniques have be en developed to approach these questions . Enzymatic or metabolic radiolabelling with 32P allows phosphoproteins to be localized or followed through sample pr ocessing and separation procedures via autoradiography on film or st orage phosphor screens. Th is type of labeling boasts extremely high sensitivity, with detecti on limits as low as only a few hundred disintegrations per minute (dpm; 1 uCi = 616.7 dpm) (Annan et al. , 2001), and selectivity for the detection of phosphorylation. In additi on, it can be incorporated into a wide array of experiments, both in vitro and in vivo . 32P labeling can be used to determine which proteins within a mixture are phosphoryl ated, to locate the specific site(s) of phosphorylation, and to quantify pho sphate incorporation at part icular sites. Classically, radiolabeled phosphoproteins are separated and identified by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophores is) or 2D (isoelectric focusing followed by SDS-PAGE) gels. Subsequent enzymatic di gestion and two dimensional separation of the radio-labeled phosphoprotein digests pe rmits the localization of phosphorylated peptides on a 2D peptide map (Boyle et al. , 1991; Sineshchekova et al. , 2004). These maps may be used in and of themselves to conduct time course a nd functional studies of phosphorylation on each peptide sequence. In addition, labeled phosphopeptides may be recollected and characterized by Edman sequenc ing or mass spectrometry to identify the peptide and locate the exact site of phosphorylati on (Roach and Wang, 1991). These methods, though tedious and time c onsuming, can provide rich information about phosphoproteins. In vivo analysis, while possible th rough metabolic labeling and

PAGE 19

5 indirect “back phosphorylation” analysis, can be complicated by slow turnover of phosphate groups, low overall phosphoprotein le vels, and the natural population of nonlabeled organic phosphate present in the cell (Moore and Sefton, 1996; Sineshchekova et al. , 2004). Recent findings also indicate that metabolic incorporation of radioactive 32P at working doses may induce DNA fragmentation, elevate tumor suppressor protein levels, and alter cellular and nuclear morphology, resu lting in cell cycle arrest or cell death (Steinberg et al. , 2003) Additionally, lab safety must be carefully considered in order to reduce personnel and equipmen t exposure to radioactive -emissions. Fluorecent identification. Recently, a fluorescent phospho-specific stain, ProQ Diamond (Molecular Probes, Eugene, OR), ha s been developed to visually locate phosphoproteins on a 1D or 2D gel without the use of radioactivity. Because the phosphoproteins are labeled post-protein isolation, in vivo , and therefore presumably physiologically relevant, phosphorylation can be directly monitored with this technique over a wide dynamic range (1 ng – 1 g) (Steinburg et al. , 2003). Combined with Sypro Ruby staining for total protein quantitati on, a relative amount of total phosphate incorporation can be calculated (Steinburg et al. , 2003), though mixed re views about this capability have been received. Because the non-covalently bound stain is easily washed off and use of radioactivity has been avoided, a wide array of conjugated analyses may be applied for downstream analysis of phosphoprotein samples. The complementary nature of this technique to downstream analys is is critical to its success, since identification and quantitation of exact sites of phosphorylation within a protein using this stain alone have not been demonstrated to date. Low to m oderate levels of nonspecific binding to non-

PAGE 20

6 phosphorylated proteins and prot eins with chemically simila r modifications have also been reported (Martin et al. , 2003), and therefore directed an alysis of individual proteins must be performed to verify phosphorylation (Steinburg et al. , 2003). In general however, this method offers a simple, fast, and global means of screening for phosphorylation in complex cellular extracts th at may be applied in both research and clinical settings. Phosphoamino acid analysis. Upon isolation of a labeled phosphoprotein, whether identified by fluorescence or radioi maging, phosphoamino acid analysis may be performed to determine the nature of the phospho rylated residues in the protein. This is accomplished through acid hydrolysis of the prot ein of interest, spot ting of the resulting sample onto a thin layer cellulose chromatogr aphy plate, and subjec ting it to high voltage 2D electrophoresis (Sefton, 1997; Yan et al. , 1998). While this procedure enables identification of the type of amino acid phosphor ylated (serine, threonine or tyrosine) it does not answer the additional questions of wh ere or when the protein becomes modified in vitro or in vivo , and therefore maintains only limited usage. Antibody detection. Antibody detection of phosphoprotei ns is another widely used technique. Phosphoproteins modified on tyrosi ne residues may be isolated from whole cell lysates by immunoprecipita tion with phosphotyr osine specific antib odies, rendering these antibodies a means of sample precon centration for phosphotyrosine systems (Moore and Sefton, 2000). While antibodies for phosphos erine and phosphothreonine have also been generated, they lack the required se lectivity and sensitivity for quantitative immunoprecipitation applications. These anti bodies instead tend to be sensitive to the amino acid sequence in the region of the phosphorylation, and therefore generally find

PAGE 21

7 utility only once phosph orylation at a particular loca tion on a particul ar protein has already been determined (M oore and Sefton, 2004; Steinburg et al. , 2003). In these instances, phosphopeptide specific antibodies for phosphoserine or phosphothreonine systems may be produced for immunoprecipita tion of particular phosphoproteins of interest. In addition, both phosphoserine/phosphothreo nine and phosphotyrosine antibodies may be used as primary antibodies for the detection of phosphoproteins that have been blotted to nitrocellulose or PVDF me mbranes down to femtomole levels (Yan et al. , 1998). Detection here, however, relies also on the sensitivity, se lectivity, and binding efficiency of a secondary antibody bearing a fluo rescent tag, or an enzyme tag that can be visualized by a colorometric or chemilumi nescent system. Steric hindrance of the phosphorylation site by native pr otein structure may also in terfere with antibody labeling (Yan et al. , 1998). Downstream analysis for the determination of the exact sites of phosphorylation on a protein, when not already known, is also complicated due to the requirement for removal of the antibody and label under relatively harsh conditions (Steinburg et al. , 2003). Although all of these techniques have played crucial roles in the development of our current understanding of protein phosphorylation, no one of these systems can address all aspects of protei n phosphorylation analysis (Whi ch? Where? When? How much?). Thanks to its versatility and re latively high sensitivity and selectivity, mass spectrometry has enabled analysis of protein phosphorylation both in vitro and in vivo , and may be used in conjunction with front end separation and/or labeling techniques to answer the broad range of phosphorylation-base d questions posed of a given system.

PAGE 22

8 Mass Spectrometry in Protein Phosphorylation Analysis Simply put, a mass spectrometer is com posed of three main components: an ionization source, a mass analyzer, and a detect or. The mass analyzer region utilizes the basic laws of classical physics to resolve th e differential motion of ions of different mass to charge ratio (m/z) in electric fields, magnetic fields, or in the absence of any type of field. Based on this very general defi nition, two fundamental requirements are introduced. First, the analyte mu st be ionized and be in the gas phase to be detected, and second, the mass analyzer region must be kept under vacuum in order to extend the mean free path of ions in the analyzer, thereby allo wing for the detection of ion motion that has been minimally perturbed by collisio ns with background molecules. A wide variety of ionization sources ha ve been designed to meet the first requirement for mass spectrometric analysis. The advent of “soft” ionization techniques, most notably electrospray ionization (ESI) (Fenn et al. , 1989) and matrix-assisted laser desorption ionization (MALDI) (Karas and Hi llenkamp, 1988), has enabled the analysis of larger, non-volatile molecules, thereby expanding the realm of mass spectrometry’s application into fields such as proteo mics and genomics. Developments and improvements in mass analyzers and vacuum systems have not only increased resolution and mass accuracy capabilities, but have also resulted in an in crease in the versatility of experiments which may be performed within the instrument itself. These experiments will be discussed further in the following sections. Ionization of Phosphorylated Peptides and Proteins Yet another challenge to pr otein phosphorylation analysis is introduced when using mass spectrometry to monitor this modification. Because the process of ionization is not a democratic one, the chemical nature of analytes in a mixture will determine the relative

PAGE 23

9 extent to which each will be detected. Th e anionic nature of the phosphate group hinders ionization in the positive mode (the most commonly used mode of operation). As a result, in the presence of the non-phosphorylat ed version of the same peptide or other non-phosphorylated compounds in the sample mixture, ionization of the phosphorylated analyte will be suppressed. Electrospray ionization In terms of electrospray ionization (ESI), which is the method of ionization used throughout the work to be presented in this dissertation, this suppression effect can be conceptualized through a basic understandi ng of one accepted theory of the ESI mechanism. The sample is dissolved in a solvent and drawn through an emitter tip in the presence of an electric field high enough to induce charge separation at the liquid surface. As charges build at the liquid-air interface at the emitter tip, the liquid meniscus takes on the form of a cone and jet (the Taylor cone) in order to most efficiently distribute these charges. Once the forces of Coulombic repu lsion exceed those of the solvent’s surface Figure 1-1: Formation of “mini” Taylor cone from ESI-produced charged droplets. [Reprinted with permission from Gomez, A.; Tang, K. “Charge and fission of droplets in electrostatic sprays.” Physics of Fluids 1994, 6 , 404-414. Copyright 1994, American Institute of Physics.]

PAGE 24

10 tension (the Rayleigh limit), sm all, highly charged liquid drop lets, with typical diameters of approximately 1 to 2 m (Wilm and Mann, 1996) assuming microliter/minute flow rate, are finally released from the end of the Taylor cone. As the solvent evaporates and Coulombic repulsion again increases to the Ra yleigh limit, “miniatur e” Taylor cones are formed on the surface of the initial dropl ets and droplet fission produces the next generation of smaller, charged droplets (Fig. 1-1) (Cech a nd Enke, 2001). This cycle of solvent evaporation, increased repulsion of th e charges, and droplet fission continues until completely desolvated ions are finally released and drawn into the mass spectrometer for analysis. General relationships between the operat ional parameters in ESI dictate the optimization of the process both globally a nd for particular types of analytes. For instance, the amplitude of the electric field necessary to initiate the ESI process is directly proportional to the surface tension of the solven t used, since higher el ectric potentials will be required to meet and exceed the Raylei gh limit of a solven t with higher surface tension. Once the ESI process has begun, the ion current leaving the emitter tip is directly proportional to the conductivity of the sample so lution (which is in turn dependent on analyte or electrolyte concen tration), the flow rate, the solvent surface tension, and the solvent permittivity. While this initial current should theoretically correspond directly to ion current measured at the detector of the mass spectrometer, ion transmission inefficiencies delinearize this relationship, and instead ion current at the MS detector relates more directly to initial droplet diameter. This trend is one of the foundations of ion suppression e ffects in ESI (Cole, 1997).

PAGE 25

11 Individual ESI droplets contain a represen tative distribution of analytes from the original sample solution, as well as a number of excess charges which are forced to the surface of the droplets by Coulombic repul sion. The surface activity of a specific analyte, which depends largely on the polarity and hydrophobicity of th at analyte, plays a predominant role in determining the extent to which it will respond to electrospray ionization. The equilibrium-par titioning model proposed by Enke et al. in 1997 describes the nature of excess charge distribution on the surface of droplets and the simultaneous preference of non-polar, hydrophobic analytes to also inhabit the surface of droplets (Enke, 1997; Cech and Enke, 2001). According to this model, polar, hydrophilic analytes will prefer the hydrophilic core of the dr oplet, where no excess charge resides. Additionally, ion transmission optic s provide analytes with only a finite time and space to reach a stage of final ion desolvation, as was alluded to earlier, and therefore ion losses on the order of four to five orders of magnitude are genera lly expected (Zook and Bruins, 1997). As a result of equilibrium-partitioning, populations of these ion losses are greatly skewed toward the polar, hydrophilic analytes (Zook and Bruins, 1997) and in this way, ionization and detection of this class of anal ytes is severely hindered. This presents a clear limitation in phosphorylation analysis, since the polar, hydrophilic nature of the phosphate group draws these compounds toward the center of ESI droplets. In addition to surface activity, the ionic nature of each anal yte in solution, as well as their gas phase basicity or proton affinity, determine both th e mechanistic pathway of ionization (charge separation, adduct formation, gas phase reaction, etc.), and the effici ency of ionization by ESI.

PAGE 26

12 The suppression of ionization of phosphorylat ed analytes in ESI can be avoided by separation of these analytes from other components which may demonstrate higher proton affinities, and therefore “out-com pete” phosphorylated compounds for the excess charge. For this reason, high pressure liquid chromatography and other front end separation techniques which are easily coupled on-line to most ESI sources have had a great impact on analysis of analytes de monstrating low ionization efficiency. The evolution of nano-electrospray ionizat ion (nESI) has also improved our ability to detect these hard-to-ionize components even when mixtures are too complex to separate completely. By reducing the ini tial droplet size of the spray through reduced flow rate and emitter tip inner diameter, nESI forces hydrophilic analytes closer to the point of ion sputtering, thereby enhancing io nization and detection of such analytes. Traditional nESI, initially demonstrated by Wilm and Mann in 1994, employs flow rates which are driven by the electrospray process itself (no solvent pumps used), reportedly in the range of 20 to 40 nL/min and resulting in predicted initial droplet diameters on the order of 200 nm (Wilm and Mann, 1996). Though achievement of such low flow rates requires off-line analysis th at is often unsuitable fo r high throughput or complex proteomic applications, efforts to decrease emitter tip diameter and flow rate on any system can significantly enhance sensitivity to ward phosphorylated analytes according to these principles. Alternative ionization methods: MALDI, ICP Other ionization methods such as MALD I and inductively coupled plasma (ICP) ionization have been used successfully to study protein phosphoryl ation. MALDI, which was already introduced as anothe r “soft” ionization technique, has benefited the field of proteomics greatly. With its slightly higher tolerance for sa lts and buffers present in the

PAGE 27

13 sample solution as compared to ESI (Beavis and Chait, 1990) and its adaptability to coupling matrix compounds with affinity cap ture media for on-tar get preconcentration and minimized sample handling (Raska et al. , 2002), MALDI offers an interesting alternative to phosphoproteomic ionization and an alysis. However, difficulty in on-line coupling of MALDI with front end separation techniques, its reduced compatibility with MS/MS analysis (since only one charge is typically imparted on each ion), and the low reproducibility associated with sample deposition procedures (resulting in the limited opportunity for quantitative anal ysis) all restrict its appli cation in this field. ICP provides a very different and extr emely sensitive means of phosphorylation analysis by detecting elemental phosphorus in a sample. Despite its promise in this field, the abundance of isobaric interferences which necessitate the coupli ng of this ionization source to instruments capable of providing mass resolving powers of greater than 1500, as well as the ubiquitous presence of environmental phosphorus that can easily contaminate samples, make ICP a somewhat less prominent techni que to date (Becker et al. , 2003). Mass Spectrometric Methods of Detecting Protein Phosphorylation Once the requirement of ionization is met, the mass spectrometer itself may be used in a variety of ways to targ et protein phosphorylation. The use of these methods depends wholly on the type of mass sp ectrometer available and the na ture of the sample to be analyzed. Characteristic mass shifts. Because the phosphate modification is a labile one, it can be unintentionally lost in the mass spectromete r to produce characteristic mass shifts of m/z 98 (positive ion mode, +1 charge state), m/z 49 (+2 charge state), m/z 32.6 (+3 charge state), and so on. In this way, a sample may be analyzed before and after

PAGE 28

14 phosphatase treatment to locate those peptide ions which shift in mass by these values (McLachlin and Chait, 2001). Instruments capable of some form of tandem mass spectrometry may also be used in this wa y by applying a relatively low fragmentation energy to initiate loss of labile modificat ions such as phosphorylation. Peptide ion masses before and after disso ciation may then be surveyed for those ions which demonstrate the characteristic mass shifts. These experiments are typical of MALDI and/or direct infusion ESI analysis, wh ere whole sample mixtures are observed simultaneously. While these types of analyses enable the identification of phosphorylated peptides, further analysis is required to locate the exact residue of phosphorylation within the peptide. Tandem Mass Spectrometry (MS/MS). A fundamental understanding of the mechanisms of ion fragmentation in genera l facilitates understa nding of the different methods of fragmentation to be introduced. Most fragmentation techniques employed in conventional mass spectrometry are ergodic proc esses which allow for the redistribution of energy throughout the ion prior to fragmentation. As a resu lt of this vibrational energy redistribution, dissociation of the amide (p eptide) bond, the weakes t bond in the peptide backbone, is the most frequently observed product of fragmentati on. These techniques include collisionally induced dissociation (CID) (Laskin et al. , 2003), sustained off resonance CID (SORI-CID) (Laskin et al. , 2003), and infrared multiphoton dissociation (IRMPD) (Little et al. , 1994). In each of these techni ques, the internal energy of ions trapped in a higher pressure collision region of the instrument is increased by either implementation of an on or off resonant radio frequency (RF) pulse, acceleration upon introduction into a higher potential field, or irradiation and subse quent absorption of

PAGE 29

15 infrared photons. The elevated internal ener gy of the ions render th em more susceptible to fragmentation as they continue to collid e with inert gas molecules infused into the collision cell of the mass spectrometer for this purpose. Internal energy is redistributed throughout the ion, inevitably resulting in dissociation of the w eakest molecular bonds. It should become clear at this point that under normal MS/MS settings a non-modified peptide ion will produce a relatively random array of fragments along the lower energy backbone bonds, which facilitate its identification and seque ncing. Conversely, a peptide bearing the labile phosphate moiety will c onsistently exhibit predominant loss of the phosphate group, rendering little information as to peptide sequence and complicating identification of the exact phos phorylation site. Still, obser vation of a characteristic fragment ion spectrum (a predominant ion at [M – (H3PO4/n)]n+ with few additional low intensity fragment ions) is used to id entify phosphorylated ions by conventional techniques such as liquid chromatogr aphy–data dependant mass spectrometry (LCMS/MS). Complete characterization of th e phosphorylation site and peptide sequence may require additional analysis. The use of high mass accuracy and high resolution instruments such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, the recently introduced Orbitrap, and to a lesser degr ee, the hybrid quadrupole time-of -flight (qTOF) instruments greatly reduces the severity of this pr oblem. The high mass accuracy of parent ion measurements using these instruments, in conj unction with characteristic mass shifts in the MS/MS spectra, can often identify the phosphorylated peptide ion from a list of possible peptide sequences in a database, as well as lessen the probability for isobaric interferences.

PAGE 30

16 MS3. Lower resolution/ mass accuracy instruments may utilize MS3 for the sequencing of a peptide ion. In this way, a pa rent ion is isolated, subjected to the first stage of fragmentation in which the pare nt ion minus the phosphate modification is produced. This ion may then be isolated and subjected to a second st age of fragmentation in which an array of fragment ions are produced which can give information on the sequence of the parent ion. Exact location of the phosphate modification may then be localized by the presence of fragment ion mass diffe rences corresponding to dehydroalanine or beta-methyl dehydroalanine, which represent the beta elimination products of phosphoserine or phosphothreonine, respectively. Recent improvements in data acquisition software enable the re searcher to perform data-dependent MS3 analysis upon recognition of the characteri stic neutral loss mass shif t in an MS/MS scan of a particular parent ion. Precursor ion scanning. Triple quadrupole and qTOF instruments offer an additional means of highly selective phosphorylation anal ysis by utilizing the lability of the phosphate modification. Firs t developed in 1993 by Carr et al. , precursor ion scanning has greatly enhanced the ab ility of mass spectrometry to study post translational modification of all kinds (Carr et al. , 1993). In these experime nts, which are performed in the negative ion mode, mass separation oc curs in the first quadrupole, followed by fragmentation in the second. Subsequently, only fragment ions with an m/z of -79, corresponding to the mass of the phosphate i on, are scanned out to the TOF region by the second quadrupole in qTOF instruments, or to the detector by the third quadrupole in triple quadrupole instruments. Based on the separating power and resolution of the first quadrupole, parent ion masses can be deduced from the tim e at which the phosphate ion

PAGE 31

17 reaches the detector. While this technique does not provide high resolution determination of parent ion mass or fragment information for peptide identificati on, it is an extremely sensitive means of decipheri ng a list of ion masses that may be targeted for datadependent MS/MS in positive ion mode. This concept may also be utilized by the continuous monitoring of the m/z -79 ion by negative mode LC-nESI-MS, followed by coll ection of the fractions rendering the phosphate specific ion. Direct infusion precursor ion scanning of each fraction to deduce parent ion mass is then followed finally by positive mode nESI-MS/MS for peptide isolation, fragmentation, sequenci ng, and site identification (Annan et al. , 2001). The development of triple quadrupole instrume nts which allow for polarity switching on a chromatographic time scale has greatly simp lified and automated such experiments. Electron capture dissociation. Though the exact mechanism of fragmentation is not yet completely understood, el ectron capture dissociation (ECD) is known to be a nonergodic mechanism of ion fragmentation. By this method, trapped ions are irradiated with low energy electrons (<7 eV ) that are absorbed into N-C (amine) backbone bonds, resulting in immediat e dissociation (10-16 – 10-15 sec, a rate which exceeds that of vibrational relaxation and energy redistributi on by orders of magnitude) of these bonds (Zubarev, 2003). In this way, labile post translational m odifications are retained on fragment ions, greatly facilita ting localization of the modifi cation. ECD is currently only commercially available in FTICR mass spect rometers, due mainly to the ICR cell’s unique ability to trap ions and electrons w ithout significantly cha nging the kinetic energy of the electrons in the re gion of interaction (Zubare v, 2003). In addition, the non-

PAGE 32

18 destructive means of FTICR detection enable s the collisional cooling and re-irradiation and fragmentation of larger peptide or protein ions for “top-down” analysis. Phosphopeptide/ Protein En richment and Separation Based on the complexity of the sample and the information sought from the experiment, phosphopeptide or protein enrichment may be necessary prior to analysis, even when front-end reversed phase liquid chromatography and/or nESI are employed. Preconcentration may be achieved by methods previously described, and/or through solid phase affinity capture techniques, includi ng immobilized metal ion affinity capture (Anderson and Porath, 1986), or metal oxide capture (Ikeguchi and Nakamura, 1997). Sample fractionation based on ion exchange mechanisms (Trinidad et al. , 2006) has also been demonstrated, as well as phosphoami no acid specific chemical modification for enrichment and analysis of phosphoryl ated sample components (Stevens et al. , 2005). Immobilized Metal Ion Affinity Chromatography Immobilized metal affinity chromatogr aphy (IMAC) was introduced in 1975 by Porath and coworkers as Metal Chel ate Affinity Chromatography (Porath et al. , 1975). Originally used for protein fractionation based on differential affinity to immobilized zinc or copper ions, the technique boasted charac teristics of both affinity and adsorption chromatography. Preferred association of phosphorylated amino acid residues to immobilized Fe3+ was later demonstrated in 1986 by Porath and Anderson (Anderson and Porath, 1986; Muszynska et al. , 1986). Today, the technique is primarily exploited as a versatile affinity capture method. Along with the purification of multi-histidine tagged recombinant proteins, the enrichment of phosphorylated proteins and peptides has evolved into one of the majo r applications of IMAC.

PAGE 33

19 IMAC columns generally consist of a sili ca, agarose, or polymeric structural support, a chelating ligand such as iminodiacet ic acid (IDA), nitriloacetic acid (NTA), or triscarboxymethyl ethylene diamine (TED), and a metal ion of choice (Chaga, 2001). The choice of chelating ligand and metal i on play important roles in experimental planning. IDA, NTA, and TED form trid entate, tetradentate, and pentadentate coordination complexes with the metal ion, respectively. While ligands with a higher degree of coordination will have increasingly higher affinity for the metal ion, a simultaneous decrease in analyte binding affin ity to the metal ion is observed due to the loss of available metal coordination sites (Gaberc-Porekar and Menart, 2001; Ueda et al. , 2003). Based on the strength of analyte-metal ion association predic ted, the researcher may chose a chelating ligand system to be st fit the needs of the application. The choice of a metal ion is undoubtedly the mo st critical step in the design of this type of experiment due to the great variabil ity in metal ion behavior with regard to different analytes and solvent systems. A classification system developed by Pearson (Pearson, 1973) provides a general direction fo r the determination of which metal ion is suitable for a particular application based on Lewis acid/base chemistry. According to this classification system, “soft” meta l ions (the Lewis acids), such as Cu+, Hg2+, and Ag+, prefer “soft” Lewis bases, such as su lfur-containing amino acid side chains. “Intermediate” metal ions, including Cu2+, Ni2+, and Co2+, favor coordination with borderline Lewis bases such as aromatic ni trogen. “Hard” metal ions, such as Fe3+, Ca2+, and Al3+, prefer binding to “hard” Lewis bases such as oxygen and a liphatic nitrogen. (Pearson, 1973; Chaga, 2001; Ueda et al. , 2003).

PAGE 34

20 While most early studies of phosphopeptide/ protein enrichment focused on the use of Fe3+, many studies have been performed to de termine the effectiveness of alternative hard metal ions for this application in term s of retention and recovery. Notably, a 1999 study by Posewitz and Tempst compared rete ntion, elution, and se lectivity of eight different metal ions (Al3+, Ga3+, Fe3+, In3+, Ru3+, Sc3+, Y3+, Zr4+) with respect to three synthesized phosphopeptides in the presence of a standard pr otein digest. Ga3+, Fe3+, and Zr3+ demonstrated maximum retention and el ution (competitive binder) capabilities, while Ga3+showed the best selectivity and pH de pendent elution properties of the three metal ions toward the three synthesized phosphopeptides (Posewitz and Tempst, 1999). The wide array of important experimental para meters that must be controlled from lab to lab has made it difficult to designate a si ngle metal ion as the optimal choice for phosphopeptide/ protein preconcentration. Regard less, to date the most success in these applications has been attributed to use of Fe3+ and Ga 3+. In addition to metal ion a nd chelating ligand selection, a number of other factors govern the association of a metal ion with its preferred Lewis base. Ionic strength of solvents used, pH, and the composition of buffers and/or detergents used in the analysis all play important roles in the effectiveness of IMAC enrichment (Ueda et al. , 2003). In general, increasing ionic stre ngth of binding and washing so lvents reduces electrostatic interactions of analytes with the chelating ligands and stru ctural support. At low ionic strength however, chelating ligands such as hard metal ion bound IDA exhibit weak ion exchange properties, which can enhance the preconcentration of phosphopeptides and phosphoproteins by providing an additional mechanism of phosphopeptide/protein retention. The pH of solutions used has a critical effect on the overall charge of the

PAGE 35

21 immobilized metal ion, as well as the charge s present on the protein or peptide binding surface. As such, pH can be modified to pr omote the desired coordi nation interactions of the metal ion with specific chemical groups on the analyte (i.e., the phosphate group). For optimal binding and retention of phosphor ylated analytes, pH of the binding and washing solutions should be maintained above the pKa of the phosphate group (1.97) (Kumler and Eiler, 1943), though below the pKa of side chain carboxylate groups (3.86 for aspartic acid, 4.25 for glutamic acid) (Dawson, 1959). Buffers, denaturing agents, and detergent additives mainly affect appli cations involving whol e protein enrichment, where protein structure and sol ubility may influence binding ca pabilities. Again, the type of application determines the qualitative requirements of the above mentioned parameters. (Sharma and Argarwal, 2001) Titanium Dioxide Enrichment The high affinity of titanium dioxide toward organic phosphate has been recognized since the late 1980s (Ikeguchi and Nakamura, 199 7). Since then, moderate efforts have been placed into optimizing th e interaction so that this property may be utilized for the preconcentration of phosphate -containing analytes. Successful binding and elution of both phospholipids and phosphope ptides have been demonstrated under acidic and then basic pH conditions, re spectively, in both off-line and on-line configurations (Ikeguchi and Nakamura, 1997; Ikeguchi and Nakamura, 2000; Prinkse et al. , 2004; Larsen et al. , 2005). To date, this technique ha s proven to be an effective and versatile alternative to IM AC enrichment of phosphorylated peptides. Recent applications include the preparation of TiO2 coated magnetic nanoparticles that may be used to selectively adsorb phosphorylated pep tides from tryptic protein digests, followed by direct anaysis of these phosphopeptidebound particles by surf ace assisted laser

PAGE 36

22 desporption ionization mass spectrometry (S ALDI) (Chen and Chen, 2005). In addition, because binding and elution is primarily pH-dependent, as opposed to the array of variable mobile phase conditi ons associated with IMAC en richment, incorporation into automated on-line analyses is greatly simplified (Pinkse et al. , 2004). -elimination/ Michael addition The identification of phosphorylated re sidues may also be accomplished through the chemical modification of phos phoamino acid residues themselves. -elimination of the phosphate group (phosphoserine and phosphothreonine only) under alkaline conditions results in the formation of an alkene group that is highly susceptible to nucleophilic attack. This transformation perm its Michael addition of a thiol-bearing (or similarly nucleophilic) compound at the previ ously phosphorylated location. The nature of the tag added by Michael ad dition is flexible and main ly dependent on the desired downstream analysis. Possibilities include biot in tags, which lend to the sensitive capture of these elements by biotin/avidin affinity chromatography, or fluorescent affinity tags, which facilitate the enrichment and enhan ced spectrometric detection of previously phosphorylated compounds (Stevens et al. , 2005). In addition to the transformation of the phos phorylation site itself into a target for affinity enrichment and detection, removal and replacement of the negatively charged phosphate group generally enhances ionization efficiency of th ese peptides in the positive ion mode, again simplifying analysis. Unfortuna tely, selectivity of the technique suffers from the ease at which any O-linked posttranslational modification will undergo elimination. For example, by these met hods, protein phosphorylation can not be distinguished from protein glycosylati on, another abundant post translational

PAGE 37

23 modification. Inconsistent or inadequate r eaction yield is another disadvantage of this technique. Cation/Anion Exchange for Phosphopeptide Fractionation The ionic character of th e phosphate group itself may be exploited through ion exchange chromatography. In this way, frac tionation by charge state may enable partial separation of phosphorylated peptides or prot eins from a complex mixture, such as a whole cell lysate. The addition of such a fract ionation step greatly simplifies analysis of complex mixtures, and enhan ces the ability to detect and characterize low abundance proteins such as those typically involv ed in protein phosphoryl ation-based signaling pathways (Trinidad et al. , 2006). Hydrophobicity-based Adsorption Techniques While reversed-phase chromatography us ing C-18-type resin is typical of proteomic separation and analysis, small, hydrophilic peptides are poorly retained on these chromatographic matrices. Phosphorylat ion can increase the hydrophilicity of a peptide considerably, especially in smaller or more acidic peptides, making retention and separation by traditional methods difficult. In these instances, alternative adsorptionbased stationary phases may be employed. These include hydr ophilic interaction chromatography (HILIC) (Alpert, 1990) , porous graphitic carbon (PGC) (Knox et al. , 1986; Vacratsis et al. , 2002), and C-18 resins with ch emistries with stronger hydrophobic interactions. HILIC, unlike normal revers ed phase chromatography, demonstrates a greater retention for hydrophilic, charged com pounds. PGC, which functions similarly to typical reversed phase chromatographic ma trices for most compounds, exhibits an additional electrostatic binding property due to the analyte-induced deformation of carbon ring electron clouds. In th is way, PGC can be used to tightly retain, recover, and

PAGE 38

24 resolve small, hydrophilic analytes such as those which may be produced post-enzymatic digestion of a phosphorylated protein. Alternatively, Poros Oligo-R3 (Applied Biosystems) is an example of a C-18 st ationary phase with high hydrophobic binding affinity. Success of this stationary phase for these types of app lications relies on the hydrophobicity of amino acids adjace nt to the phosphoamino acid. Protein and PTM Quantitation by Mass Spectrometry While the relative measurement of cha nges of mRNA levels within a given cell state, or states, have become routine expe riments via microarray analyses, a functional understanding of cellular phenotype s requires the quantitative an alysis of proteins, since mRNA and respective protein le vels do not necessarily corr elate at a given time point (Gygi, Rochon et al. ,1999). Regardless of the mRNA level corresponding to a given protein, it is the cell’s stimulus to translate that mRNA into pr otein that dictates when and to what extent the protein is expressed. Moreover, protein function may be arrested until, or changed by, various types of post transl ational modification. The past decade has witnessed the escalating success of mass spectrometry in th e elucidation of functional proteomics. Though the ability to quantita te by mass spectrometry is not an innate characteristic of the technique due to variable ionization effici encies of different analytes, many methods incorporating stable isotope la beling and label-free quantitation have been developed to overcome these inherent limitation s, and utilize the unique characteristics of mass spectrometry for protein and PTM quantitation. Relative Quantitation In many applications, it is the changi ng level of protein expression or post translational modification brought about by a particular physiol ogical, environmental, or pharmaceutical stimulus that is the desired information. In these cases, differential

PAGE 39

25 isotopic labeling with stable isotopes of a control and variable cell state enables the subsequent pooling and MS analysis of the tw o cell states simultaneously. In this way, changes in protein expression or levels of post translational modification may be directly evaluated. To date, a number of chemical labeling schemes have been developed for relative quantitation of changes in protein ex pression or post transl ational modification. These include isotope coded affinity tags (I CAT) that modify cy steine residues (Gygi, Rist et al. , 1999), acid labile isotope c oded extractants (ALICE) (Qui et al. , 2002), isobaric tags for relative and abso lute quantitation (iTRAQ) (Ross et al. , 2004), methyl esterification of carboxylic groups (Goodlett et al. , 2001; Ficarro et al. , 2002), lysine specific labeling (Peters et al. , 2001), N-terminal labeling (Munchbach et al. , 2002; Lill, 2003), 18O incorporation during enzymatic cleavage (Yao et al. , 2001), and differential mass mapping or label-fr ee quantitation (Ruse et al. , 2002; Bondarenko et al. , 2002). Metabolic incorporation of stable isotopes into a cell line via either growth in 15N enriched media (Oda et al. , 1999), or stable isotope la beling by amino acids in cell culture (SILAC) (Ong et al. , 2002), are extremely efficien t methods of differential protein labeling which allow for a broad scope of downstream analysis. These specific methods will not be discussed in detail he re. Instead, focus will be placed on the considerations that must be made in order to attain useful information from labeling experiments. First and foremost, choice of an appropriate label is crucial. Ideally, a label would demonstrate good reaction efficiency, be universally effective to all peptides in a mixture, have no effect on comparative chromatographic retention so that peptide pairs experience the same conditions (percent organic of m obile phase, chemical background, etc) upon

PAGE 40

26 ionization. The label must also provide ample mass separation and resolution of differentially labeled peptides (to avoid overl ap of isotope peaks) , have no differential effect on ionization efficiency, and demonstrat e no differential (or otherwise) effect on dissociation efficiency. Add itionally, the label would be equally applicable to cell culture, tissue culture, or clinical sample sets . Clearly, designing a label of such diversity is an enormous task, however based on the sp ecific experimental design, an appropriate and effective label may be chosen to provi de the desired information (Lill, 2003). In order to measure biologically significan t values a variety of controls must be employed. First, a linear dynamic range (LDR ) and lower limit of quantitation (LLOQ) for the instrument to be used must be es tablished by a series of standard curves. Subsequently, internal standards must be a dded into, or identified from, the original sample mixtures in order to control for di screpancies in sample handling and labeling efficiency. In the case of post translational modification analysis, differences in initial protein concentration due to sample collec tion variability, or di fferences in protein expression between two cell states , must also be controlled for by the internal standards. Internal standards may also be used to ensu re that measurements are being made within in a linear dynamic range. Absolute Quantitation When an absolute molar quantity of a part icular protein of in terest is desired, traditional quantitative analysis strategies may be applied. Generally, a peptide, or set of peptides, from the protein of interest are synt hesized with stable isotope labels so that they may be used as inte rnal standards (Barnidge et al. , 2003; Gerber et al. , 2003). Standard curves may be generated in order to determine the ESI response factor for the analyte of interest, the LDR, and the analyte’s specific LLOQ based on its ESI response

PAGE 41

27 factor and the standard deviation of blank measurements. The heavy labeled standard peptide may then be spiked into the biol ogical sample at a kn own quantity, and by its ratio with the non labeled peptide from the sa mple, used to determine a quantity of the sample peptide. Since the standard is spiked into the sample late in the sample preparation, however, extreme caution must be taken to avoid error or sample loss in previous sample processing steps. For this reason, additional in ternal controls that will account for each step of sample processing, such as those described earlier, may be necessary. Collectively, with the proper choice of internal standard/c ontrols, method of separation, ionization method, and instrumentation, relativ ely accurate quantitation is possible by mass spectrometry. Summary The purpose of this chapter was to introdu ce the fundamental principles behind the work to be described throughout the rest of this dissertation. A variety of mass spectrometric and associated methods, including sample enrichment, liquid chromatography, ionization, and quantitation, have all been described as they apply to protein phosphorylation and proteomic analys is. Chapter 2 will provide in-depth descriptions of the instrume ntation utilized for the present work. In chapter 3, the analysis of Limulus polyphemus Myosin III in vivo phosphorylation, and the changes induced in phosphorylation of this protei n in response to multiple physiological or environmental stimuli will be demonstrated. In chapter 4, a quantitative determination of the preferential binding charac teristics of different member s of the 14-3-3 family of signaling proteins from Arbidopsis thaliana to phosphorylated target peptide sequences is

PAGE 42

28 presented. Finally, chapter 5 will attempt to unite the presented work and provide broad conclusions regarding the im pact of these projects.

PAGE 43

29 CHAPTER 2 INSTRUMENTATION Three types of mass spectrometers were ut ilized for the work to be described, namely, a Bruker Daltonics BioApex II 4.7 T FTICR-MS, a ThermoFinnigan LCQ Deca 3D ion trap MS, and an Applied Biosyste ms QSTAR XL hybrid quadrupole TOF MS. Each instrument demonstrated vastly different but complementary operational capabilities and limitations, making access to all three instruments a considerable advantage in experimental planning. Each of these instruments will be described in attempt to emphasize the unique characteris tics of each, and the scope of their applications to the present work. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Thanks to its ultra high mass resolution a nd mass accuracy capabilities (Amster, 1996; Marshall et al. , 1998), as well as its impressive experimental versatility, FTICRMS has found an ever growing realm of app lication. Recent improvements to front end ion guides and electronics have prompted a marked increase in commercial sales of FTICR mass spectrometers over the last decade, making the unique and impressive capabilities of this type of MS more accessible than ever. FTICR mass spectrometers are generally com posed of an ionization source, a series of differentially pumped ion transfer optics and ion guides (multipoles, linear ion traps, ring electrodes, skimmer cones, steering plates, etc), and an ICR cell within the bore of a superconducting magnet. The extraordinary cap abilities of FTICR-MS with regard to high resolution and mass accuracy are directly attributed to the unique mechanism of ion

PAGE 44

30 Figure 2-1: Configuration of trapping, excitation, and de tection plates in ICR cell. trapping, mass analysis, and detection. Ions are trapped in the ICR cell by a potential placed across two plates set perpendicular to the incoming ion beam and magnetic field axis (Fig. 2-1). At this point, ions exhi bit three types of inherent motion: trapping oscillations, magnetron motion, and cyclotr on motion. Trapping oscillations are an artifact of the ion’s translat ional kinetic energy upon entering the cell. Linear motion is converted into a simple harmonic oscillati on upon application of the DC trapping plate voltages. While the trapping potentials produce a restoring fo rce that restricts the ions’ motion along the magnetic field axis (z-axis), the harmonic nature of this motion repels them in the perpendicular plane (xy plane) . Simultaneously, the magnetic field repels ions toward the center of the cell, and in turn the competition of these forces results in the initiation of magnetron motion. Steps are often taken prior to excita tion and detection to dampen these two types of low frequency motion in order to cool ions closer to the center of the cell, thereby enha ncing resolution upon analysis (Amster, 1996; Marshall et al. , 1998).

PAGE 45

31 Figure 2-2: Ion cyclotron mo tion. Motion is derived from the cross product of the magnetic field and the radial velocity component of th e ion. Direction of the motion is dependent upon the charge of the ion. (adapted from Marshall et al. , 1998) The third type of ion motion, cyclotron motion, is the foundation of FTICR-MS. Cyclotron motion occurs as a result of the magnetic field’s effect on the ion’s radial velocity component, producing a force that is perpendicular to the magnetic field axis. This force sets the ion into a periodi c, circular motion whose frequency ( c) is dependent upon the strength of the magnetic fi eld (B), the mass of the ion ( m ), and the charge on the ion ( z ), as shown in Equation 2-1. By keeping the magnetic field constant, this cyclotron frequency ( c) can be directly related back to th e ion’s m/z (Amster, 1996; Marshall et al. , 1998). Equation 2-1: In order to detect the cyclotron frequenc y, the orbital radius of an ion must be increased such that it may induce an im age current across the detect plates. Amplification of the cyclotron orbital radius is achieved by application an oscillating electric field that is in resonance with the specific cyclotron frequency of the ion across two opposing excitation plates that run parall el to the magnetic field axis. Usually, a range of resonant frequencies is applied to th ese plates at a set amilitude. As each ion of a specific m/z value “feels” its characteristic resonating freque ncy, its cyclotron radius is Bz 2 m c =

PAGE 46

32 increased to a degree proportional to the amplitude of the applied field (Vp-p) (Equation 22). In this way, either a specific ion population may be excited by a defined RF excitation energy for detection, or all ions within a predefined range may be excited by the application of an RF sweep at a constant amplitude for the simultaneous detection of all ions in a mixture (Amster, 1996; Marshall et al. , 1998). Equation 2-2: For optimal detection, the applied RF amplitude is set to excite all ions to a radius in which the coherent ion packets pass close t o, but do not collide with, the detect plates, two oppositely aligned plates also in parallel with the magnetic field axis (Fig. 2-1). As the ion packet passes each of th e detect plates, an alternati ng current is induced with a frequency identical to that of the characteristic cyclotron frequency of the ion. This image current is amplified and converted fr om the time domain signal to a frequency domain via a Fourier transform, which can th en be directly convert ed to a m/z spectrum (Amster, 1996; Marshall et al. , 1998). The high resolution of this measurement is a direct result of the high magnetic field strength, the accuracy at which frequenc y can be measured, and the number of measurements taken per scan. The time it take s an ion to make one complete revolution in its cyclotron orbit is multiple orders of magnitude lower then the observation or scan time (milliseconds to seconds). For example, an excited ion of 100 m/z in a 3 tesla magnet travels approximately 30 kilometers per second (Marshall et al. , 1998). In a 4.7 tesla magnet, such as the one used in this study, the same ion would have a cyclotron frequency of approximately 114 kHz, and woul d therefore induce an image current to be detected roughly 114,000 times per second. This leads to an extremely high degree of Vp-pTexcite 2dB rpost-excite =

PAGE 47

33 precision and resolving power that can play an important role in accurately distinguishing the isotope patterns of high mass, multiply-c harged ions, leading to their proper mass assignment, or in accurately determining sub-nominal mass differences between lower m/z ions. In addition, as the magnetic fiel d strength is increased, the spread of frequencies that corresponds to a set range of mass/charge va lues is increased, therefore increasing the difference between frequencie s corresponding to any two consecutive mass/charge values. In addition to the measurement of an i on’s m/z, multiple opportunities exist for the analysis of fragment ions. Multip le stages of mass analysis (MSn) can offer abundant structural information about th e parent ion in question. More over, the number of possible stages of ion fragmentation in an FTICR-MS is theoretically unlimited, as fragmentation events inside the ICR cell are separated tem porally as opposed to spatially in many other instruments. Ion dissociation may take place via a number of routes, including collisionally induced dissociati on (CID) (Amster, 1996; Marshall et al. , 1996; Laskin and Futrell, 2003), sustained off resonance CI D (SORI-CID) (Laskin and Futrell, 2003), and infrared multiphoton dissociation (IRMPD) (Little et al. , 1994; Tonner and McMahon, 1997; Flora and Muddiman, 2001; Flor a and Muddiman, 2002; Hakansson et al. , 2003). The benefit of in-cell fragmentation methods resides in the fact that all ions other than the ion of interest can be selectively overexcited and ejected from the cell prior to fragmentation, isolating the pr ecursor ion to be dissociated and ensuring the origin of observed fragment ions. For CID and SORI-CID, the ion of interest is isolated, after which a low pressure pulse of a neutral collisi on gas such as nitrogen is infused into the cell (Amster, 1996, Marshall et al. , 1998, Laskin and Futrell, 2003). A low amplitude RF

PAGE 48

34 voltage that is either in res onance with the analyte ion (CID) or just off-resonance relative to the analyte ion (SORI-CID) is then applied across the two excite pl ates (Amster, 1996; Marshall et al. , 1998, Laskin and Futrell, 2003). The purpose of this RF pulse is to increase the ion’s kinetic energy (and in turn the orbital radius ), increasing the probability of collision with a neutral gas and simultaneously rendering it more susceptible to dissociation upon collision (Laski n and Futrell, 2003). Cleav age of the weakest bonds in the ion is induced as a result, giving rise to product ions th at are subsequently excited and detected as described above (L askin and Futrell, 2003). Becaus e excitation sets the ions off-axis however, ions must be collisionally “re-cooled” after the second detection event in order to induce additional fragmentat ion with minimal signal decay (Tonner and McMahon, 1997; Hakansson et al. , 2003). The third method of ion dissoci ation, IRMPD, involves an infrared lase r irradiation pulse of specified pulse length and power in order to produce the desired excitation and fragmentation. Though the mechanism of ion exc itation is drastically different from that of CID, rapid energy redistribution th roughout the bonds of ions renders the fragmentation pathways very similar (Little et al. , 1994). Because the power and pulse length of the laser irradiati on event can be accurately and precisely controlled however, bond-stability discrimination is ach ievable by this technique to some extent. This enables a stepwise approach to specifically cleaving bonds of increasing st ability by adjustment of laser power and pulse length (Flora a nd Muddiman, 2002). In a ddition, ion activation and dissociation occur on-axis with the laser beam, reducing the tota l number of cooling and pumping delays required in the instrume nt method since no ion “c ooling” or collision gas pumping delays are required between disso ciation and detection. This presents a

PAGE 49

35 significant advantage over typical CID experiments when multiple stages of fragmentation are desired. Furthermore, because the frequency of the carbon dioxide infrared laser (10.6 m) is in resonance with the stretchi ng frequency of the P-O bond (9.6 – 11 m), a specifically attenuated laser pulse can selectively cleave pho sphate groups from trapped ions at very low irradiation times (Little et al. , 1994; Flora and Muddiman, 2002). This results in characteristic mass shifts (79.966 Da) of only those peptide ions which originally possessed the phosphate mo iety (Flora and Muddiman, 2001) FTICR-MS was utilized in this work fo r its ability to simultaneously survey mixtures of peptides that were closely rela ted in size and chemical nature with a high degree of mass accuracy and resolving power. Methods were developed to characterize binding preferences of a mixture of syntheti c phosphopeptides to a family of enzymes called the 14-3-3s, which will be discussed in detail in chapter 4. Quadrupole Ion Trap Mass Spectrometry A Thermo LCQ Deca 3D ion trap mass spectrometer was also used in these studies for multiple applications. Like FTICRMS, 3D ion traps are tandem-in-time mass spectrometers, implying the use of a single ion trapping region for each stage of analysis. Unlike FTICR-MS, ions are trapped and ejecte d for detection by careful control of a set of RF voltage sweeps (as opposed to freque ncy sweeps at a set voltage or amplitude) across three electrodes, a centr al ring electrode and two end cap electrodes. Externally formed ions are transferred to the trap by a se ries of ion optics; in this case, a heated metal capillary for the desolvation and transm ission of ESI formed charged droplets, an offset tube lens and skimmer cone to focus ions and block transmission of neutrals, a

PAGE 50

36 square RF-only quadrupole for ion transmi ssion through regions of differential pumping, and an RF-only octapole for final tran smission of ions into the trap. Figure 2-3: Configuration of el ectrodes in 3D quadrupole ion trap. (http://www.spectroscop ynow.com/ftp_images/msprim_spark_f5c.jpg) Within the trap, ion collection and movement are governed by the high RF voltage (kV), or drive voltage, placed on the ring electrode . This RF voltage confines ions in the center of the trap as they os cillate between the relatively neutral end-cap electrodes and the ring electrode with the changing phases of the applied RF. A much lower amplitude (0 – 5 V) and lower frequency RF voltage, refe rred to as the resonant excitation voltage or ‘tickle’ voltage, is pla ced on the end-cap electrodes 180 out of phase from each other. The magnitude and frequency of this voltage can be varied throughout a scan function in

PAGE 51

37 order to isolate ions, initiate CID, and in crease resolution during mass analysis. The addition of helium at approximately 1 mtorr into the trap aids in extension of the linear dynamic mass range, as well as improvement of resolution due to col lisional cooling of the ion cloud closer to the central axis of the trap (Burlingame, 2005; March and Todd, 2005). After ion trapping, specific ions of interest may be isolated for dissociation. In this case, the RF voltage on the ring electrode is incr eased such that the i on of interest aquires a “q” value (defined by Mathieu equations, see Fig. 2-4) of 0.83. A large range of high Figure 2-4: The Mathieu stab ility diagram describing ion st ability in a quadrupole ion trap. The “q” value is a function of th e RF voltage while the “a” value is a function of the DC voltage. 3D ion tr aps are typically operated in RF only mode, such that a ramping of the RF voltage moves ions along the x-axis, increasing that ions “q” value until it passes out of the region of stability. (http://www.matrixscience.com/ help/ion_trap_main_help.html) a = 4e U m 0 2 2 -2e V m 0 2 2 q = U = DC voltage V = RF voltage e = charge of ion m = mass of ion = angular frequency = t / 2

PAGE 52

38 amplitude RFs, excluding a small belt of frequencies surrounding that which resonates with the ions bearing the 0.83 “q” value, are th en applied to the end-cap electrodes. This results in an over-excition and ejection of all ions but the ions of interest. The ring electrode RF voltage is then decreased in orde r to move the ions of interest back to a lower “q” value (~0.25) such that upon fragment ation, an even distribut ion of smaller and larger product ions may be stably trapped. The small range of resonant frequencies which were excluded from the is olation pulse are now applied to the end cap electrodes at an amplitude sufficient to increase the kine tic energy of the ions of interest without overexciting them out of the trap. Th e energy imparted does, however, induce fragmentation upon collision with helium atoms (Burlingame, 2005; March and Todd, 2005). Ejection of precursor and/or product ions for detection is accomplished by linearly elevating the ring electrode RF amplitude, thereby increasing the “q” value of all ions proportionally. Each ion of specific m/z ratio is ejected from the tr ap sequentially; lower m/z ions first, followed by higher m/z ions. Simultaneously, a resona nt excitation voltage is applied (to the end-cap elec trodes) at a constant frequenc y which correlates to a high “q” value, resulting in incr eased resolution. These ions are detected and signals amplified by a conversion dynode and electr on multiplier (Burlingame, 2005; March and Todd, 2005). An extremely important feature of the Th ermo LCQ Deca 3D ion trap instrument used in these studies is automatic gain cont rol (AGC), which manages the number of ions collected in the trap prior to further stages of analysis. Because of the relatively small trapping volume, an overfilling of the trap will lead to space charge effects that decrease

PAGE 53

39 resolution and mass accuracy. To avoid these affects, a 10 ms prescan is taken prior to each analytical scan in order to survey the flux of ions entering the tr ap at any point in a chromatogram. The accumulation time of the anal ytical scan is then adjusted based on the measured flux in order to collect a pred etermined optimal number of ions to be measured in each analytical scan. The tota l ion chromatogram displayed in turn is actually an inverse function of the accumulation time of the an alytical scan, since longer collection times will be required of low concen tration ions, while shorter collection times will be required of high concentration ions. This feature enhances sensitivity of lower abundance ions of interest while avoiding space charging effects on mass accuracy and resolution, thereby doubling th e linear dynamic range of the instrument (March and Todd, 2005). Quadrupole ion traps have contributed greatly to the incr eased use of mass spectrometry in proteomics. The versatile na ture of their operation, small size, relative low cost, ability to perform MSn, and the ease at which they can be precisely controlled by computer automation have made them a standard in most biological mass spectrometry labs. The ability to operate th ese instruments in a “data-dependent” mode enables the collection of a massive amount of information-rich data per sample, while targeted analysis can allow for sensitive measurement of low abundance compounds of qualitative or quantitative inte rest. Improvements to instrument control software are currently expanding the realm of experiments which may be conducted by the researcher at will. For the purpose of this research, the LC Q Deca was used for the verification of in vivo phosphorylation sites in Limulus polyphemus Myosin III. This work will be

PAGE 54

40 discussed in detain in chapter 3. In add ition, this instrument was used to develop a method for future automated semi-quantitativ e analysis of 14-3-3 target phosphopeptide mixtures, which will be desc ribed further in chapter 4. Hyrbid Quadrupole-Time of Flight (qTOF) Mass Spectrometry Unlike FTICR and 3D ion trap instruments, the quadrupole – time of flight (qTOF) mass spectrometer is a tandem-in-space hybrid instrument which utilizes two different types of mass analyzers. Modeled after th e triple quadrupole tandem mass spectrometer, the qTOF utilizes the superior mass selection and ion transmission capabilities of the first two quadrupoles, while replacing the thir d scanning quadrupole with an orthogonally aligned TOF mass analyzer. Traditional trip le quadrupole instruments, renowned for their ability to efficiently perform MS/MS e xperiments for structural elucidation, employ the first quadrupole as a mass selector, the se cond quadrupole as a co llision cell for CID, and the third to mass-analyze product ions. The need to scan the third quadrupole at a slow enough rate to obtain unit resolution ac ross a complete mass spectrum of daughter ions greatly limits the sensitivity of these instruments however. Figure 2-5: Schematic of the Applied Biosystems QSTAR XL hybrid qTOF MS.

PAGE 55

41 By replacing the third quadrupole with a TOF analyzer, a complete full scan or fragment ion spectrum can be detected in pa rallel at the maximum resolution afforded by the TOF spectrometer. The general equa tion which describes TO F mass separation is: m/z = 2 e E s ( t/d )2 where ‘ m/z ’ is the mass to charge of an ion, ‘E ’ is the extraction pulse potential, ‘ s’ is the distance across which E is applied, ‘ d ’ is the length of the field free drift zone, and ‘ t ’ is the measured “time of flight” of the ion. Re solution in a TOF instrument is dependent on the final thickness of an ion p acket as it approaches the de tector and the length of the flight path. Optimizing ion beam quality (min imizing spatial and velocity distributions) prior to acceleration into the TOF through collisional cooling or focusing, and addition of an ion “mirror” or reflector (to correct for initial spatial deviations and motion along the TOF axis while effectively doubling the length of the flight tube) enhance resolution considerably (Chernushevich et al. , 2001; Burlingame, 2005). Because a TOF analyzer is not a scanning device, duty cycle, and therefore sensitivity, is also enhanced by subst itution of the third quadrupole with a TOF spectrometer. The necessity to modify the continuous ion beam delivered by the RF-only quadrupole into discrete, pulsed ion packet s for TOF analysis however, presents a challenge in preserving the gained sensitivity. In the system used in this work, this challenge is met by a “Q2 pulsing” function. He re, ions are briefly trapped in the second quadrupole region (the collision cell) by intr oduction of an exit lens upon which an trapping voltage can be applied. The volta ge on this lens is dropped for 10 to 50 milliseconds in order to pulse a packet of ions into the pulsar region of the orthogonal TOF. A delay time is then set to coordinate the release of the ion packet from the second

PAGE 56

42 quadrupole with the TOF acceleration pulse, ther eby reducing sample loss and increasing duty cycle. Because ions of different mass/ch arge will reach the pulsar region at different times, the ion release pulse value and ion release delay must be tuned for optimal transmission at the targ eted mass range of the quadrupoles (Chernushevich et al. , 2001; Burlingame, 2005). An added benefit of such a configuration of analyzers lies in the ability to exploit either MALDI or ESI sources with little modification to in strument parameters, despite the differences in initial ion en ergies and beam characteristics. An additional quadrupole, q0, placed at the front end of the instrument provides a region of collisional cooling of newly formed ions, damping the ion velocities to near-thermal values and focusing ions to the center quadrupole axis (perpendicular to the TOF axis). Whet her ions are formed continuously (as in ESI) or in pulsed plumes (as in MALDI), beam quality is improved drastically prior to injection into the orthogonal TOF. This additional stage of focusing at q0 aids in eliminating sourceinduced energetic aberrations from ions prior to injection into the final mass analysis region, and enhan ces resolution of measurements in the TOF by reducing initial motion along th e TOF axis prior to acceler ation. For ions formed by MALDI in particular, the transformation of th e ion plume into an ion beam decouples the TOF acceleration pulse from the ionization la ser pulse, offering a dditional control of optimization parameters (Chernushevich et al. , 2001; Burlingame, 2005). Generally speaking, the qTOF enab les high mass accuracy (typically 5 ppm) and high resolution (10,000 to 15,000) mass analysis in both full scan and MS/MS modes of operation. Its tolerance for different ion s ource configurations en ables coupling with front end separation devices, such as HPLC, a nd software control of operating parameters

PAGE 57

43 and experiment types give it some of the ve rsatility of triple quadrupole instruments (Chernushevich et al. , 2001; Burlingame, 2005). In the applications to be described in the following chapters, the instrument is operated in information dependent acquisition (IDA) mode in order to identify peptide ions eluting from an in-line integrated HPLC system for the purpose of protein identification and post tran slational modification (P TM) characterization and quantitation. A survey scan of eluting i ons is first acquired by operating all of the quadrupoles in RF-only mode. In this wa y, ions are cooled, focused, and transmitted from the source to the TOF region for full s can mass analysis. Information dependent MS/MS spectra can be obtained by using Q1 to select a major parent ion from the survey scan for fragmentation, q2 for CID of the pare nt ion and transmission of the daughter ions into the pulsar region of the TOF, and the TOF for fragment ion mass analysis. Final detection is achieved by means of a four-anode multichannel plate detector with a timeto-digital converter, which offers high sensitiv ity (detection of singl e ions) and resolving power over a relatively large mass range. It is worthwhile to note here that th e mechanism of inducing CID in the qTOF (high potential acceleration into the high pre ssure region of q2) offers an advantage for phosphopeptide characterization over CID frag mentation in the quadrupole ion trap (resonance excitation). Resonance excitation induced CID usually yields a predominant ion representing the neutral loss of the phosphate moiety, thereby providing little additional sequence information. Higher ener gy CID in the qTOF yields a more even distribution of fragment ions, facilitating phosphope ptide identification and characterization. For this r eason, and due to the ability to obtain high resolution and

PAGE 58

44 mass accuracy, information dependent LC-MS/MS data, this instrument was utilized for the identification of phosphorylation site s in recombinant Limulus Myosin III (Battelle et al. , 2004; Kempler et al. , submitted), as well as the relative quantitation of changes in in vivo phosphorylation at the identified sites in response to circadian rhythms. This work will be described in detail in Chapter 3.

PAGE 59

45 CHAPTER 3 SITE SPECFIC EFFECTS OF THE CIRCADIAN CLOCK ON LIMULUS MYOSIN III PHOSPHORYLATION The function of Limulus polyphemus Myosin III (LpMyoII I), a 122 kDa protein found in Limulus photoreceptors, is unknown. This protei n originally stirred interest in the Battelle laboratory (Whitney Laboratory, University of Florid a, St. Augustine, FL) when it was identified as a protein that became phosphorylated in Limulus eyes in response to signals from a circadia n clock (Edwards and Battelle, 1987) Circadian signals reach Limulus eyes via a well-characterized group of efferent neurons with cell bodies located in the brain and axons that project out the optic nerves and synapse onto the photoreceptors (Battelle, 2002). As depicted in Fig. 3-1, the clockdriven depolarization of these efferent ne urons at night releas es octopamine onto photoreceptors (Battelle et al. , 1982). This biogenic amine activates a G protein-coupled receptor that is linked to adenylyl cyclase. Activation of adenylyl cyclase stimulates an increase in intracellular levels of cAMP in photoreceptors (Kaupp et al. , 1982) and initiates cAMP-dependent signaling cascades , one of which invol ves the phosphorylation of LpMyoIII (Battelle et al. , 1998). Overall retinal function is directly influen ced by this circadian efferent input, or clock input. As clock input to the eye is elevated in the evening and remains active throughout the night (Barlow, 1983), the sensit ivity and responsiveness of the eyes to light increases.

PAGE 60

46 Figure 3-1: Schematic represen tation of the signaling cas cade responsible for circadian clock-driven changes in visual function in Limulus . The circadian clock responsible for these cha nges is located in the Limulus brain (left). The gray bar (center) represents th e photoreceptor membrane. Clock activated efferent neurons projecting from the brain to the eyes release the biogenic amine octopamine (OCT) onto photoreceptors and the OCT activates G-protein coupled receptors (G) that activate adenylyl cylase (AC). Activation of AC elevates intracellular cAMP which in tu rn activates cAMP-dependent protein kinase (PKA). PKA phosphorylates substrate proteins (adapted with permission from author from Battelle, 2002). Many specific structural and functional effects of this input have been characterized, including the i nduction of changes in photorec eptor and pigment cell shape and changes in pigment migration (Barlow et al. , 1980; Keir and Chamberlain, 1989). Clock input also increases the magnitude of the depolarization response recorded from photoreceptors per photon of light absorbed and simultaneously decreases the number of spontaneous depolarizations recorded from phot oreceptors in the dark. Thus, clock input to the eye at night increase s the overall signal-to-noise ra tio (Kaplan and Barlow, 1980). Additionally, clock input primes light driven process such as pigment migration and

PAGE 61

47 membrane shedding, processes which do not occur without night-time circadian clock input (Chamberlain and Barlow, 1979; Chamberlain and Barlow, 1984). A complete understanding of how clock input exerts its diverse effects on photoreceptor functions requires an in-depth kno wledge of the proteins that are affected by this stimulus. The iden tification of a 122 kDa phosphopr otein that demonstrated enhanced phosphorylation in response to circadian input st imulated further characterization of the protein sequence a nd structure. The protein was cloned and sequenced, revealing a C-terminal myosin domain and an N-terminal kinase domain (Battelle et al. , 1998). This evidence classified the pr otein as a class III Myosin (one of the unconventional myosins within the myos in superfamily). Additional evidence suggested it was unique among metazoan m yosins in that it became phosphorylated within its myosin motor domain (Battelle et al. , 1998). Phosphoamino acid analysis of the protein identified serine as the sole phosphorylated residue (E dwards and Battelle, 1987), and primary sequence analysis revealed three putative cAMP dependent protein kinase (PKA) phosphorylation sites (S796, S846, S926) in or near a known actin binding region in myosins called loop 2 (Battelle et al. , 1998). Also within the myosin domain of the protein are sequences associated with ATP binding and myosin-type conformational changes (which occur upon ATP hydrolysis), a putative calmodulin binding domain, and the TEDS site (E618, an acidic amino acid located at a position highly conserved among metazoan myosins that is found to be replaced by a phosphoamino acid in proteozoans) (Battelle et al. , 1998). All of these observations l ead to hypotheses concerning LpMyoIII function.

PAGE 62

48 Elucidating these functions re quires an understanding of th e specific interactions of LpMyoIII. Myosins that are molecular mo tors bind to actin and hydrolyze ATP to produce the conformational ch anges that permit the myosin to move along actin filaments. Actin binding and ATP hydrolysis were therefore assumed to be important functions of LpMyoIII. Actin co-sedimentation assays have demonstrated that LpMyoIII does in fact bind to actin (Battelle et al. , 2004). The myosin domain of LpMyoIII also has an ATP binding site, however while the ATP binding site is mostly conserved, an arginine residue thought to be critical for ATP hydrolysis ha s been replaced by a histidine (H487), suggesting that LpMyoIII ma y not hydrolyze ATP (Battelle et al. , 1998). Indeed, ATPase assays revealed that LpM yoIII does not hydrol yze ATP (Battelle et al. , 2004). Taken together, these finding suggest that Lp MyoIII is an actin binding protein but not a motor. The identification of PKA phosphorylation si tes in and near loop 2 of the myosin domain by LC-MS/MS analysis of recomb inant LpMyoIII led to a hypothesis that phosphorylation modulates the affinity of LpMyoIII for actin. Studies of other unconventional myosins have revealed that th e net charge of loop 2 is an important determinant of actin binding affinity (Furch et al. , 1998). Specifically, site directed mutagenesis studies of other myosins have de monstrated that an increase in the number of positively charged residues within loop 2 in creases the binding affinity of myosin toward actin (Furch et al. , 1998). Conversely, it can be extr apolated that an increase in negative charges in the same region would re duce actin binding affinity. Since it is known, as described above, that circadian efferent input to Limulus photoreceptors leads to the elevation of cAMP in photorecepto rs and in turn the PKA phosphorylation of

PAGE 63

49 LpMyoIII, it has been hypothesized by the Batt elle laboratory that th e circadian-regulated phosphorylation of specific residues in and/or near loop 2 regulates the affinity of LpMyoIII for actin. Preliminary immunocytochemical localiza tion of LpMyoIII and actin within the photoreceptor in the presence and absence of cl ock input supports th is hypothesis (Fig. 32). The distribution of LpMyoIII i mmunoreactivity in cross sections of Limulus lateral eyes which had received normal clock input wa s compared with that in eyes that were deprived of this input. All ey es were fixed in the dark and at night when the clock input is active. In eyes that received normal cl ock input the concentration of LpMyoIII over the actin-rich photosensitive membrane was re producibly lower than that in eyes deprived of the clock input (Battelle et al. , 2006). This immunocytochemical evidence is consistent with the idea that clock input modul ates the affinity of LpMyoIII for actin via phosphorylation in the regi on of actin binding. In the work to be presented in this ch apter, it was verified that the sites of phosphorylation identified in expressed recombinant LpMyoIII were indeed phosphorylated in native LpMyoIII extracted from the animal itself. Relative changes in phosphorylation at particular sites between LpMyoIII from eyes which had received clock input and those which had been deprived of clock input were then examined. The goal here is to determine which, if any, of the potential phosphorylati on sites in and near loop 2 show changes in phosphorylation levels in response to endogenous clock input to the eyes. These findings will permit additional biochemical studies in which site-directed mutagenesis of recomb inant proteins and in vitro actin binding assays will be used to test the effects of phosphorylation at these speci fic sites on LpMyoIII ac tin binding affinity.

PAGE 64

50 Figure 3-2: Immunostaining of myosin III and actin in cross sections of lateral eye ommatidia. Eyes were dissected and fixed in the dark at about midnight. Panels A and B show stained sections of eyes that received normal clock input (+ clock input).. Panels C and D show st ained sections of eyes that had been deprived of clock input by cutting the lateral optic ne rve one week prior to the experiment (clock input).. The asteri sk shaped structures defined by actin staining in panels A-D represent the photosensitive membrane, the rhabdom (Rh). (reprinted from Battelle et al. , 2006 with permission of IOVS, copyright ARVO 2006) The schematic shows the major visible components of an ommatidium cross section. (adapted with permission from author from Battelle, 2002) (Battelle et al. , 2006) Methods Animals and Standard Reagents Adult Limulus collected from the Indian Ri ver near Melbourne, FL were maintained at the Whitney Laboratory in co ntinuously flowing, na tural seawater held between 18 C and 20 C and under natural diurnal il lumination provided through a skylight in the aquarium room. Unless othe rwise specified, reagents were obtained from either Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO).

PAGE 65

51 Lateral Optic Nerve Sections, Lateral Ey e Extraction, and LpMyoIII Purification At least one week before colle cting the tissue, the lateral optic nerve to the right eye of each experimental animal was cut as desc ribed previously (Edwards and Battelle, 1987 et al. , 1990). On the day of the experiment, an imals were moved at dusk into a natural seawater tank located in a dark room, and the animals were maintained in the dark. Between 11 PM and midnight, lateral eyes were dissected from the animals under infrared illumination. In all subsequent steps, eyes with cut optic nerves ((-) clock input) were processed separately from those with intact optic nerves ((+ ) clock input); each sample contained eyes pooled from three or four animals. Sample s were homogenized in the dark in homogenizing bu ffer containing phosphatase inhib itors, protease inhibitors, and reducing agents (Sineshchekova et al. , 2004), and homogenates were centrifuged for 20 minutes at 120,000 x g in an Airfuge (Beckma n Instruments). At this point, samples were moved into the light, and supernatants were filter concentrated using 30 kDa cut-off membranes (Millipore). SDS P AGE buffer (4x) was added at a volume calculated to be 1/3 the remaining volume of th e supernatant. The two different samples were loaded onto different 7.5% Tris-glycine SDS-PAGE ge ls poured from the same pre-polymerized mixture and run at 150V for 1 hour. Gels were stained with Coomassie Blue for 5 minutes with shaking, and were destained fo r approximately 5-6 hours in a 10% glacial acetic acid (HAc), 10% methanol (MeOH) solution. LpMyoIII Digestion The stained bands corresponding to Myosin I II were excised from the gel, cut into 1mm by 1mm cubes, and destained with gent le shaking in 50% acetonitrile (ACN), 50 mM ammonium bicarbonate (ABC). This destain solution was refreshed every 15 minutes until gel pieces were clear. The ge l pieces were dried by addition of 100% ACN,

PAGE 66

52 removal of ACN, and speed vac evaporati on of the remaining liquid. Reduction of disulfide bonds was accomp lished with addition of 45 mM DTT for 30 min at 55 C. Reduced cysteines were alkylated with 100 mM iodoacetamide for 30 minutes at room temperature and in the dark. This soluti on was removed and gel pieces were washed vigorously with 50 mM ABC containing 50% AC N. Gel pieces were finally dried by addition of 100% ACN, removal of ACN, a nd speed vac evaporation of the remaining liquid. Approximately 200 ng of trypsin in 50 mM ABC was added per single lane of gel band. Digestion was allowed to proceed overnight at 37 C. Peptides were extracted by incubating gel pieces in a solution of 50% ACN and 5% formic acid until the gel pieces began to turn white. The supernatants were then removed, dried completely, and esterified as described below. Alternatively, for label-free an alysis, the supernatants were dried to near completion and resuspended in a solution containing 5% ACN and 0.1% HAc. Methyl Esterification – Differential Labeling Anhydrous methanol was sparged with nitr ogen for 5 minutes prior to use. The two labeling solutions (light and heavy) were prepared immediately prior to use in a well ventilated area by addition of 45 l of thionyl chloride to 1 ml of either anhydrous d0methanol (CH3OH) or d3-methanol (CD3OH). Completely dried peptide digests were removed from the speed vac and the appropria te labeling solution was added immediately (1 ml solution per 8 single lane gel bands). Tubes were blanketed with nitrogen and sealed, sonicated for 10 min, and allowed to stand at room temperature for 2 hours. Reaction mixtures were then dried to near completion in a speed vac and labeled peptide

PAGE 67

53 digests were resuspended in a solution containing 5% AC N and 0.1% HAc. Samples to be compared were pooled appropriately. Figure 3-3: Schematic of both label-free and differe ntial labeling methods. LC-MS/MS analysis A portion of this work was conducted us ing a Thermo LCQ Deca quadrupole ion trap MS in line with a 5 cm x 75 m inner diameter (i.d.) Pepmap C18 5 m/ 300 capillary column (LC Packings ) or a self-packed 10 cm x 75 m i.d. Altima 5 m/ 300 capillary column. A home built HPLC system with a 1/30 pre-column split was used to deliver a 60-minute gradient from 5% to 50% mobile phase B (mobile phase A = 0.1% HAc, 0.01% TFA, 3% ACN; mobile phase B = 0.1% HAc, 0.01% TFA, 95% ACN) at a flow rate of 12 L/min. For initial identification e xperiments, parent ion scans were followed by four data-dependent MS/MS scan s. For targeted analysis, one datadependent MS/MS scan and two to three dedi cated MS/MS scans followed the parent ion scan. Spectra from all experiments were converted to DTA files, and the MASCOT search engine was used to search data within the NCBI (National Center for Biotechnology Information) database. In these searches, the taxonomy specified was metazoa, trypsin was identified as the cleavage enzyme, and carbamidomethyl was

PAGE 68

54 defined as a fixed modification. S/T phosphoryl ation and oxidation (M) were selected as variable modifications. Mass to lerances were set to 2 Da for MS and 1 Da for MS/MS. To locate eluting phosphopeptides in the base peak chromatogram, extracted ion chromatograms were generated by searching for the neutral loss of the phosphate group in multiple charge states. For targeted experiments, extracted ion chromatograms were constructed by selection of the appropriate s can filter and includi ng the mass of the ion representing the neutral loss of the phosphate group in the extraction criteria. Other studies were conducte d using an Applied Biosys tems QSTAR XL MS with an Ultimate Capillary HPLC syst em (LC Packings) flowing at 180 L/min with a 1/100 pre-column split. This integrated system delivered a 90-minute gradient from 3% to 40% mobile phase B (mobile phase A & B as described above) through a 15 cm x 75 m id, 5 m/300 PepMap C18 capillary column. All samp les were run in triplicate. During the analysis of label-free samples, three to four blanks were run between the different sample sets to prevent carryover. One survey s can followed by 1 to 3 information dependent MS/MS scan were performed and data were searched using MASCOT with the settings given above, but with mass tolerances set to 0.3 Da for MS and 0.3 Da for MS/MS, and with methyl esterification (DE and C-term) as a variable modification when appropriate. The first two isotope peaks of the average mass spectrum for each ion of interest were integrated for quantitative analysis. Differential-Labeling Data Interpretation Extracted ion chromatograms were generate d from the base peak chromatogram for both lightand heavy-labeled versions of all peptides studied. Mass spectra were averaged over the region of the chromatogr am where each lightand heavy-labeled set

PAGE 69

55 eluted. The first two isotope peaks of each isotopic cluster were integrated and the resultant areas were summed, as demonstrated in Fig. 3-4. These area values from each run were then summed to obtain a total area va lue for each peptide per experimental set. For each peptide, the total summed area of th e set of peaks corresponding to the intact optic nerve, or (+) clock, cell state was then divided by the corresponding summed area from the optic nerve sectioned (ONS), or ( -) clock, cell state. This calculation, which resulted in the ‘raw’ (+) clock/ (-) clock ratio, was applied to each studied phosphopeptide and to four control peptides. All control peptides were non-modified peptides derived from LpMyoIII that contained at least one acidic residue. Control peptides were analyzed to control for any differences in retinal tissue collection, tissue homogenization, digestion, labeli ng, and any other discrepancie s in sample handling prior to pooling. This normalization step also controlled for any differences in LpMyoIII expression between the two sets of eyes. Figure 3-4: Representative cal culation of (+) clock to () clock ratio via differential labeling method.

PAGE 70

56 The four control peptide ratios were averag ed to obtain the normalizing factor. The standard deviation, 95% confidence interval surrounding this normalizing factor, and relative 95% confidence interval was also determined from the four control peptide ratios. Raw phosphopeptide (+) clock/ (-) cloc k ratios were normalized with the average control peptide ratio and the error introdu ced by the method for each experiment was reported as the normalized (+) clock/ (-) clock value multiplied by the relative 95% confidence interval. Tables 3-1 through 3-4 demonstrate these calculation on a single representative set. Final (+) clock/ (-) clock ratios re presented the calculated change in phosphorylation at each site in response to cir cadian clock input. Three experimental sets were averaged in order to obtai n this final value, and the erro r reported in this final value was the 95% confidence interval determined from the averaged values and represented biological variation. In order to monitor reaction efficiency, fi nal data sets were searched using MASCOT with methyl esterification added as a variable modification. This modification to MASCOT search parameters f acilitated identification of any detectable non-labeled or inefficiently la beled myosin peptide product. Table 3-1: Representative determination of total peak areas for two phosphopeptides for both the (+)clock and (-)clock cell state, and subsequent calculation of raw (+)clock/ (-)clock ratios.

PAGE 71

57 Table 3-2 continued Table 3-2: Representative dete rmination of total peak areas for four control peptides in the (+)clock and (-)clock ce ll states, and the subsequent calculation of control (+)clock/ (-)clock ratios. Table 3-3: Error analysis applied to control peptide (+)clock/ (-)clock ratios. Table 3-4: Normalization of phosphopeptid e raw (+)clock/ (-)clock ratios and error propagation from control peptide erro r analysis to final values.

PAGE 72

58 Label-Free Data Interpretation Again, extracted ion chromatograms were c onstructed to visualize the elution of each peptide of interest. Mass spectra we re averaged from the regions of the chromatogram where each peptide eluted. The first two isotope peaks from each isotopic cluster were manually integrated, and areas from each peak within a set were summed. Peak areas from each run were summed to yield a total experiment area for each phosphopeptide studied as well as six control peptides (described earlier). For each phosphopeptide and control peptide, total areas from the cell state that received clock input (the (+) clock state) we re divided by the corresponding peptide total area from eyes which received no clock input (the (-) clock state) to yield a raw (+) clock/ (-) clock ratios. The (+) clock/ (-) clock ratios for all of the control peptides were averaged and the relative 95% confidence interval calculated. Raw phos phopeptide (+) clock/ (-) clock ratios were normalized with the average contro l (+) clock/ (-) clock ratio to correct for discrepancies in sample collection, hom ogenization, digestion, and instrumental performance. The relative 95% confidence interval calculated from the control peptide ratios, which provided a gauge error introduced by the method, was used to determine an expected 95% confidence interval for each final phosphopeptide (+) clock to (-) clock ratio. Results Verification and Identification of in vivo LpMyoIII Phosphorylation Sites Three phosphorylation sites; S-796, S-841, a nd S-846, were identified in previous mass spectrometric analyses of recombin ant LpMyoIII that had been phosphorylated in vitro with PKA (Battelle et al. , 2004). These same thr ee phosphorylation sites and phosphorylated S-926 were identified in an alyses of endogenous LpMyoIII using the

PAGE 73

59 Thermo LCQ Deca ion trap MS (Fig. 3-5 A-D). Of these sites, S-796, S-846, and S-926 are putative PKA phosphorylation sites, while S-841 is a predicted substrate for protein kinase C (PKC) phosphorylation. Interestingl y, S-926 was identified only in LpMyoIII from the Limulus lateral optic nerve (LON) (Fig. 3-5 D). It was not found in recombinant LpMyoIII (Battelle et al. , 2004) or endogenous LpMyoIII extracted from the lateral eye. S-796 and S-926 are both low stoichiometry phos phorylation sites, and as a result both required targeted analysis using the ion trap for identification. MS/MS spectra generated from the targeted experiments demonstrated the characteristic predominant neutral loss fragment ion of both phosphopeptides (M+2H-H3PO4), in addition to one or more additional fragment ions. As mentioned in Chapter 2, the predominant neutral loss of labile post translati onal modifications in a quadrupole ion trap MS following resonant excitation provides significant evidence for th e characterization of modified peptides. Regardless, the relatively poor quality of th ese MS/MS spectra warra nted verification of these results. Using the LC-qTOF-MS sy stem, phosphorylation at S-796 was supported by observation of co-eluting peaks that matche d the +2 and +3 charge state of this phosphorylated, tryptic peptide with high mass accu racy (~5 ppm for the +2 charge state) (Fig. 3-5 E). MS/MS of the peptide cont aining S-926 derived by qTOF-MS confirms ion trap assignment of phosphorylation at this site (Fig. 3-5 F). Interestingly, all of these phosphorylation sites fall within or near loop 2 of the myosin motor domain, a known actin binding region of myosins.

PAGE 74

60 Figure 3-5: MS/MS verification of four si tes in LpMyoIII phosphorylated in vivo. A) RSpSIQENMLLPER, identified by neutra l loss filtering on ion trap; B) AIFSSENPSPFLSpSPR, identified by neut ral loss filtering on ion trap; C) KVpSYDATDLVK, identified by target ed analysis on ion trap; D) RIpSFVDFLNR, identified by target ed analysis on ion trap; E) KVpSYDATDLVK, accurate mass alignmen t of +2 (upper panel) and +3 (lower panel) charge states eluting at same retention time after HPLC-qTOFMS for site verification; F) RIpSFV DFLNR, accurate mass identification of +2 charge state (upper pa nel) and MS/MS of this pe ptide (lower panel) by qTOF-MS for site verification. A

PAGE 75

61 Figure 3-5 Continued B C

PAGE 76

62 Figure 3-5 Continued EF D

PAGE 77

63 Validation of Quanti tation Methods The methyl esterification reaction chosen for differential labeling was tested for reaction efficiency by performing the reac tion on a simple mixture of synthetic phosphopeptides. Before and after reaction spect ra demonstrate a complete mass shift of all peptide ions equiva lent to the addition of the mass of one methyl group (Fig. 3-6). No non-reacted peptide byproduct was detected. To ensure equal labeling efficiency with both regular, d0-, and deuterat ed, d3-, methanol, aliquots of tryptic peptides from recombinant LpMyoIII were labeled with d0or d3-methanol. Samples pooled in a 1:1 and 1:2 ratio were analyzed by HPLC-M S/MS. The ratios of the non-oxidized, phosphorylated peptide containing S-846 (RSp SIQENMLLPER), as well as control peptides VLPLYGDQTAVK, SDNPPHVFAVADR , and YYSEEYLSR were compared to their expected values. Average ratios of these peptides in both sets were within approximately 20% of their calculated values, th us validating use of this labeling scheme. Figure 3-6: Methyl esterification reaction efficiency. A simple mixture of synthetic phosphopeptides labeled and analyzed via direct infusion ESI-FTICR-MS. No evidence of non-labeled byproduct was detected.

PAGE 78

64 Additionally, all experimental data files were searched using MASCOT (as described in the methods section) in order to identify a ny non-reacted or incompletely reacted peptide byproduct from LpMyoIII tryptic digests. No evidence was found for the incomplete peptide labeling over the c ourse of this work. The label free method for quantitation was validated by creating a standard sample that mimicked the biological samples to be an alyzed. In particular, four solutions of a bovine serum albumin (BSA) digest were spiked with variable quanti ties of two synthetic phosphopeptides so as to create standard so lutions with four uni que phosphopeptide to Figure 3-7: Schematic of standard samp le preparation scheme (upper panel) and validation of label free relative quantitation method (lower panel). In schematic, P = phosphopeptide; A,B,C = BSA tryptic control peptides.

PAGE 79

65 BSA digest ratios. The solutions were analyzed four times by LC-MS/MS and the signals corresponding to the phosphopeptides and three control BSA peptides were integrated. One sample set was chosen as the “control” set and si gnal intensities from this set were compared to all other respective phosphopeptid e and control peptide signal intensities. Average control peptide ratios were determined for each combination of sample comparisons (sample 1 to 2; sample 3 to 2; sample 4 to 2) and these values were used to normalize phosphopeptide ratios. Figu re 3-7 shows the final measured ratios plotted against the theoretical ratios cal culated from knowledge of the actual molar quantities of components in the solutions. A sl ope close to 1 with y-intercepts close to 0 was obtained for each phosphopeptide, indicatin g a close correlation of the measured andcalculated values. These findings validat e this method for quantifying differences in phosphorylation levels of a single protein obtain ed from two different biological samples. Changes in Levels of Phosphorylation in Response to Circadian Efferent Input (Clock Input). Differential isotopic labeling of LpMyoIII extr acted from lateral eyes that received or were deprived of clock input was used to quantify the influence of clock input on relative levels of phosphorylati on at each site. Recall that clock input to an eye is eliminated by cutting that eye’s optic ne rve (see Methods). Using the differential labeling method, phosphorylation ch anges could be measured onl y at the most intensely phosphorylated sites, S841 and S846. Final (+) clock/ (-) clock ratios obtaine d using the differential labeling approach were 0.96 +/0.27 and 1.97 +/0.28 for S841 and S846, respectively. Errors represent the 95% confidence interval and describe the de gree of biological vari ation. These ratios indicate the factor to whic h phosphorylation levels change at these sites in response

PAGE 80

66 Figure 3-8: Normalized (+) clock to (-) cl ock ratios calculated from three independent animal sets via differential labeling. Error bars represent the 95% confidence interval determined from 4 control peptides in each experimental set, indicating the magnitude of erro r introduced by the method. Table 3-5: Raw (+)clock/( -)clock ratios, average norma lization ratios, relative 95% confidence intervals, and normalized (+)clock/(-)clock values for S-841 and S-846 from each experimental set. to clock input. Ratios were calculated from three independent labeling experiments performed with three different sets of an imals (Fig. 3-8), each set containing tissue pooled from 3-4 animals. Furthermore, the values determined for each group of animals represent a sum of at least three independent LC-MS/MS runs. During the analyses of samples collected in January 2006, the LpMyoIII from each set of eyes was labeled in both configurations (d0labe led (+) clock, d3labeled (-) clock; d3labeled (+) clock, d0labeled (-) clock) to test , once again, whether the labeling step contributed to the final

PAGE 81

67 ratio. For this experiment, values obtain ed from both labeling configurations were similar and thus raw data were summed to dete rmine a single set of values for this animal set. A label-free approach was also empl oyed to quantify relative changes in phosphorylation. This approach wa s selected in order to 1) reduce the number of steps in sample preparation, and hopefully increase ove rall sensitivity of the experiments as a result, and 2) ensure that the identified tr ends observed with the differential labeling method were in fact the result of a biological variable (clock input) and not an artifact of the labeling procedure. These assays produced results consistent with data from the labeling experiments (Fig. 3-9). Average re lative 95% confidence intervals determined from control peptide ratios for differential la beling and label-free techniques were 13.1% and 6.0%, respectively. Figure 3-9: Comparison of average results from differential labeling and label-free experiments. Error bars represent the average 95% confidence interval determined from control peptides from each experimental set. This error indicates the magnitude of error introduced by the method.

PAGE 82

68 Using the label free method, changes in le vels of phosphorylation at S-796 were also measured. Though only two label-free anal yses based on two different groups of animals were completed, an average (+) clock/ () clock ratio at this site was calculated to be 2.13. As with the differential labeling method, each group of animals tested by labelfree assays consisted of LpMyoIII obtained fr om eyes pooled from three animals. Here again, the (+) clock/ (-) clock ratio of S-796 for each animal set was determined from the summed integrated peak areas from at least three independent LC-MS/MS analyses. The ability to identify and quantita te the relative changes in levels of phosphorylation at S796 using the label-free method is attributed to greater sample recovery using this method compared to the differentia l labeling method. Extensiv e sample drying is often associated with sample loss. As such, the label-free assay’s elimination of repeated sample drying steps that were required for e fficient methyl esterification, but associated with sample loss, is a clear advantage to this method. After combining data obtained from assa ys performed with both the differential labeling and label-free methods, the (+) cl ock/ (-) clock phosphorylation ratios for S841and S846 are, respectively, 0.95 +/0.15, and 1 .93 +/0.22 (Fig. 3-10A). Individual (+) clock/ (-) clock ratios for S-796 determin ed from two label-free analyses are also presented (Fig. 3-10B), demonstrating a cons istent increase in phos phorylation at S-796 in response to clock input. No cha nge in the level of phosphorylation at S926 was measured in response to clock input, as no phos phorylation at this site was detected by either method in LpMyoIII extr acted from the lateral eye.

PAGE 83

69 Table 3-6: Raw (+)clock/( -)clock ratios, average norma lization ratios, relative 95% confidence intervals, and normalized (+)clock/(-)clock values for S-796, S841, and S-846 from each set. Figure 3-10: Final (+) clock to (-) clock ra tios of three sites phosphorylation sites, indicating the degree to which phosphoryl ation changes in response to clock input. A) Average values for the tw o loop 2 phosphorylation sites, S-841 and S-846, determined from five experiment s. Error bars represent the 95% confidence interval of the ratios obtaine d from each experiment, indicating the degree of biological variation. B) Tw o individual determinations of (+) clock/ (-) clock ratio at S-796. Erro r bars represent the 95% confidence interval determined from six control peptide ratios from each experimental set, indicating the magnitude of e rror introduced by the method. Discussion This study had two major goals: 1) to verify LpMyoIII phosphorylation sites in endogenous protein, and 2) to measure change s in levels of phosphor ylation in response to clock input. Verification of these s ites was accomplished by standard HPLC-MS/MS

PAGE 84

70 methods. In order to accomplish the second goal, a quantitative method needed to be developed such that the numerical values de termined by this method accurately reflected endogenous biological changes; in this case, that of changing levels of protein phosphorylation in response to a natural stimulus. Method Development for Measuring Changes in Phosphorylation Typical quantitative analys es using mass spectrometry are aimed at determining absolute concentrations of a certain analyte in different samples. Measurements are based on the analyte’s MS-response compared to that of a known quantity of a ‘heavy’ stable isotope-labeled internal standard whose response vs. con centration have been established. A peptide’s response factor de scribes the slope of th is established linear relationship. With knowledge of each peptide’s response fact or, absolute concentrations can be calculated from the observed signal inte nsity. These calculated concentrations can be subsequently compared between different sa mple sets to determine differences in the amount of peptide in each sample (Gerber et al. , 2003; Barnidge et al. , 2003). Such a method could have been employed to achieve the second goal of this work, however this would have necessitate d the synthesis of h eavy-isotope labeled internal standards for each expected phosphorylated peptide, as well as each of the ‘control’ LpMyoIII peptides. Additionally, th e determination of ESI-response factors for each peptide would also be required prior to each analysis. Because we wanted to measure a factor of change in phosphorylation of a peptide from one cell state to the next, and also needed to quantitate changes in a num ber of control peptides in order to correct for different quantities of protein present, a direct MS-signal comparison method was chosen. Typically, extracted ion chromatogram s are integrated for quantitative analysis. However, since extracted ion chromatograms generated for these experiments were based

PAGE 85

71 on a 0.4 amu mass window surrounding the monosot opic ion of interest in the full scan, signal contributed from additional isotope peaks was not incorporated into the integration. This was a disadva ntage when measuring changes in low intensity ions since the mass of these peptide ions dictates that a significant percentage of the total ion counts be distributed to the second isotope peak. For this r eason, the first two isotope peaks were integrated from the averaged mass spectrum itself, and the areas determined by these integrations were summed to yi eld a final area value for each peptide. The summation of areas corresponding to th e first two isotope peaks, as opposed to all detectable peaks, was done to ensure equal representation of both ion populations being compared. Theoretically, the number and relative intensities of isotope peaks observed for the same peptide from different cel l states should be identical. However, the detection limit of the system used as well as the ratio of the peptide’s abundance between the two cell states determines whether or not the same number of isotope peaks is actually observed and can be integrated with the same accuracy (Fig. 3-11). As a result, integrating only the first two isotope peaks en sures that the difference in signal intensity is determined from the same number of isot ope peaks from each cell state. Since two peaks are required to determine the identity of a peptide on the instrument used, by our standards, two peaks could always be integrat ed to generate a fina l value representative of the ion population. This precaution en sured a conservative and accurate measure of change in phosphorylation. Comparisons of the two methods used for quantitation, differential labeling by methyl esterification and labe l-free relative quantitation, re vealed a clear advantage in label-free analyses. Only the two mo st intensely phosphorylated sites, S841 and S846, were

PAGE 86

72 Figure 3-11: Justification for integration of first two isotope peaks as a more accurate means of ratio determination. Represen tation of a ‘light’ isotope labeled (P) and ‘heavy’ isotope labeled (P*) set of phosphopeptide peaks from a differential labeling experiment. Red a rrows represent the baseline based on 3 different noise levels (NL#1, NL#2, and NL#3). The peaks associated with P in this representation were given arbitr ary intensity values. The value of the first isotope peak in the P* set was give n an estimated intensity value, and the values for the following isotope peaks were calculated such that they regressed by the same factor as those in the P set. A P/P* value for the sum of all peaks assuming an infinitely low detection limit in which all peaks are recorded (Total P/P*) represents the mo st accurate measurem ent of this ratio of change. P/P* values were also calc ulated from the sums of the first two isotope peaks only, as well as the sum of all isotopes detected from P or P* at each limit of detection. The P/P* valu e derived from the summed intensities of the first two isotope peaks only dem onstrated the best correlation to the total P/P*. detected following differential labeling a ssays. Only one of these two sites, S846, demonstrated changing levels of phosphoryla tion in response to clock input. Upon switching to label-free anal yses, phosphorylation at S796 was detected consistently in both

PAGE 87

73 cell states, allowing relative qua ntitation of this site in re sponse to clock input. As a result, S796 was found to be regulated by clock input. The limitation to the sensitivity of the differential labeling by methyl esterificat ion method is attributed to sample loss during additional sample drying steps required for efficient labeling. Unfortunately, since for label-free analysis samples are not pool ed and blanks are required in between analysis of samples fr om different cell states, instrument time required is at least tripled. S till, the greater sensitivity of the label-free method makes it a clear preference in analyses of low level post-translational modifications. Identification of in vivo Phosphorylation Sites in Endogenous LpMyoIII. The identity of four phos phorylation sites in endogenous LpMyoIII have been verified: S796, S841, S846, and S926. Three of these sites, S796, S841, and S846, were previously identified by HPLC-MS/MS in recombinant LpMyoIII after in vitro PKA phosphorylation (Kempler et al. , submitted). The reason(s) for why phosphorylation at S926 was not identified in the analysis of r ecombinant LpMyoIII is unclear, since it is a putative substrate for PKA based on consensus se quence correlation. It could be that the recombinant protein is folded differently fr om native protein extracted from the lateral optic nerve (LON), making the site unavaila ble for phosphorylation in the recombinant protein. Failure to detect phosphorylated S926 in LpMyoIII extracted from the LE could reflect a real biological difference between LpMyoIII located in the lateral eye and the lateral optic nerve. Alternatively, it is possibl e that this is an artifact of the different protein levels in these tissues, combined with the sensitivity limits of the instrument used. Comparison of base peak chromatogram ma ximum ion counts from LON analyses to

PAGE 88

74 base peak chromatogram counts from previ ous corresponding LE analyses offers a crude indication that the amount of LpMyoIII present in LON extracts is far greater than that in the LE, an observation congruent with prev ious Commassie staining analysis. As a result, identification of phosphorylation at S926 only in the LON might be an artifact of lower overall LpMyoIII concentrations in the LE. Future enhancements in method sensitivity may enable the semi-quantitative an alysis of phosphorylation at this site in the LE as well. Additional studies are required to determine th e biological relevance of S926 phosphorylation in endogenous LpMyoIII extracted from the LON, and the alleged lack of phosphorylation at this site in r ecombinant LpMyoIII and endogenous LpMyoIII extracted from the LE. Measurement of Site Specific in vivo Changes in Phosphorylation at PKA Sites in Response to Circadian Clock Input – Assigning Function to Phosphorylation? Of the four phosphorylation sites iden tified in endogenous LpMyoIII only two showed a significant change in their level of their phosphorylation in the LE in response to clock input in vivo, S-796 and S-846. Both sites, demonstrated a roughly two fold increase in phosphorylation in response to cl ock input relative to samples from eyes to which no clock input was received. It should be emphasized that these two-fold changes were observed with both differe ntial labeling and label-free te chniques. Both sites are putative PKA sites that were first identified as phosphorylat ion sites in analysis of recombinant LpMyoIII after in vitro PKA phosphorylation. Inte restingly, levels of phosphorylation at S-841, a putative PKC site, showed no change in response to clock input, an observation that suggests the cloc k-driven changes in phosphorylation observed are specific. Current observa tions confirm the results of previous back phosphorylation studies demonstrating the global PKAdependent phosphorylation of LpMyoIII in vivo in

PAGE 89

75 response to clock input (E dwards and Battelle, 1987 et al. , 1990). The critical additional information provided by the current st udy, which determined the exact PKA phosphorylation sites that ar e regulated by clock input in vivo , permits the generation of hypotheses regarding the physiological conseq uences of the phosphorylation event. Significance of Phosphorylation at S-796 and S-846. One of the PKA sites influenced by clock input, S846, is localized within loop 2 of the myosin motor domain of LpMyoIII, a known actin binding region of other myosins. The other site which demonstrated cloc k-induced increase in phosphorylation, S796, is located immediately upstream of loop 2, although still within a region of the protein that may influence actomyosin interaction (J.R. Se llers, personal communi cation). Manstein et al. (1998) established a direct relationship between the net number of positive charges in loop 2 and the strength of the myosin – ac tin interaction, such that as the number of positive charges increases, so does interaction strength. Based on these results, we propose that a reduction in net charge at loop 2 via local phosphorylation will decrease myosin – actin interac tion strength. Since th e phosphorylation of S796 S846 becomes elevated at night in response to clock input, it appears that one of the major impacts of clock input on LpMyoIII is to reduce its affinity for actin at night. This hypothesis is consistent with the recent finds of Battelle et al. (2006) that the concentration of LpMyoIII at the actin-rich rhabdom is reduced at night in response to clock input (See Introduction and Fig. 3-2). These studies provide the first example for a second messenger kinase-mediated phosphorylation of a myosin in or near loop 2 in vivo , and they reveal what might be a novel mechanism for a phosphorylation-based modul ation of the myosin – actin complex. The unambiguous identification of phos phorylation sites and clock-regulated

PAGE 90

76 phosphorylation sites of endogeous LpMyoIII now permits further investigation into the impact of phosphorylation on th e function of the protein usin g site-directed mutenagenic studies. Possible Functional Consequences of Ph osphorylation at S-796 and S-846 for the function of Limulus photoreceptors.. The functional consequences of the proposed circadian clock-induced reduction in affinity of LpMyoIII for actin could be many and diverse. One known function of myosins is to stabilize actin. If in response to night-tim e, clock-induced phosphorylation the affinity of LpMyoIII for actin is lower during the night than it is during the day, one might expect the stability of actin in the ac tin-rich core of the photosensitive membrane to be lower at night than dur ing the day. There is one welldocumented process in the LE that is thought to involve the destabiliza tion of the actin core of the photosensitive membrane. The process is called “transient rhabdom shedding”. Transient rhabdom shedding is triggered by the light of dawn but will not occur without night-time input from the circadian clock. It involves a rapid breakdown and rebuilding of the actin-rich photosensitive memb rane and results in the rapid synchronous internalization of some of this membrane which contains visual pigment (Fig. 3-12) (Sacunas et al. , 2002). Transient shedding is thought to enable a ra pid desensitization of the photoreceptor upon first light, converting th e eye to its day-time lower sensitivity state. The phosphorylation of LpMyoIII in l oop 2 might play an important role in the priming of this event by dest abilizing the actin core of the photosensitive membrane during the night. Reduced concentration of LpMyoIII associated w ith the actin in the rhabdom at night (Fig. 3-2), might also perm it light activated actin -severing proteins

PAGE 91

77 Figure 3-12: Ommatidia cross sections fr om a lateral eye which had received optic nerve input (control) and one to whic h the optic nerve had been severed (ONS). These images were taken just before dawn and 3 hours after dawn, and were immunostained for opsin, a pr ominent membrane protein associated with the photoresponse and localized at the photosensitive rhabdomeral membrane. Post-dawn membrane sh edding is clearly observed in the ommatidia which had received normal ef ferent input, whereas no shedding is apparent in the ommatidia to which efferent input had been blocked. This highlights the requirement for normal ni ght time circadian efferent input in preparing the lateral eye for this light -activated dawn shedding event. better access to the actin, thereby permitting it’s dawn breakdown. Additional experiments are needed to test these hypotheses. If the circadian clock does indeed gov ern myosin – actin interaction via phosphorylation in and around loop 2 of LpMyo III, the induced migration of LpMyoIII away from actin in the rhabdomeral membrane could be the factor which destabilizes the structured actin filaments, weakening memb rane structure and “priming” dawn shedding events as a result. Additional experiments mu st be completed in order to confirm these results in support of our hypothesis.

PAGE 92

78 Future Directions A third set of optic nerve sectioned animals will be analyzed by the label-free method in order to verify the magnitude of change in phosphorylation at S-796 in response to clock input. To further investig ate the functional effect s of changing levels of LpMyoIII phosphorylation in response to circadian efferent input, the binding affinities of several mutated recombinant Lp MyoIIIs will be assayed. In these mutated forms of LpMyoIII, the identified phosphoryl ation sites will be substituted with acidic residues, which will mimic the local charge state modification produced by phosphorylation, or neutral residues that canno t be phosphorylated or negatively charged. These experiments will help test the hypothesi s for a phosphorylation-based regulation of actin binding through local charge state modification. In addition, phosphopeptide specific LpMyo III antibodies can now be produced to study the LE spatial distributions of LpMyoIII phosphoforms along with the nonphosphorylated form. These experiments done ove r a time course can both spatially and temporally resolve intracellular localization and migration of LpMyoIII with respect to other proteins, and in response to circadian efferent input as well as other stimuli. Environmental light is another importan t extracellular regulator of photoreceptor function that could possibly effect change s in part though LpMyoIII phosphorylation. Light has three general func tional consequences on invertebrate eyes, namely the activation of Ca2+ channels resulting in excitation , adaptation of the eyes toward sustained light input, and st ructural changes to the photosensitive membrane (Dabdoub and Payne, 1999). Protein kinase C is a key play er in these light-activ ated events (Jinks et al. , 1996; Minke et al. , 1990). Specifically, the pharm acological activation of PKC results in functional effects which mimic that of natural light input. It is currently

PAGE 93

79 unknown whether photoreceptor specific LpMyoIII plays a role in these light-activated signaling cascades, however the presence of a putative PKC site in loop 2 of LpMyoIII makes this a reasonable speculation. Results of Preliminary Assays of Ligh t-Regulated LpMyoIII Phosphorylation Preliminary experiments using the labelfree relative quantita tion method described here were conducted to determine effect s of light input on levels of LpMyoIII phosphorylation. All experimental parameters were as already described aside from preparation of the animals. For these experi ments, animals were allowed to experience natural dawn and then were brought into room light (50 W/cm2) for 30 minutes. This enabled the eyes to transition normally fr om a night-time to a day-time state. One eye on each animal was then tightly patc hed with duct tape and black plastic to permit it to dark adapt while the other eye was exposed to 1 mW/cm2 light for the remainder of the day. Light-adapted eyes were removed at 5 pm in the light, while darkadapted eyes were removed at approximately the same time under infrared illumination. Both sets of eyes were processed and an alyzed by the label-fr ee methodology described earlier in this chapter. Surprisingly, the consensus PKC site with in loop 2 did not show an increase in phosphorylation levels in respons e to light. Instead, the PKA site within loop 2 was the only site detected to demonstrate a measur able change (Fig. 3-13). Though described thus far as a consensus PKA site, this site ha s also been found to also be a major site for autophosphorylation (Battelle et al. , 2004) indicating the existence of multiple mechanisms for phosphorylation at this site.

PAGE 94

80 This result is clearly a surprise and not yet understood. The experiment will be repeated with modification. There is suspici on that since the eyes were removed late in the day, the circadian efferent neurons ma y have already begun firing resulting in enhanced phosphorylation of S-846. To isolat e changes in levels of phosphorylation to strictly light input, eyes can simply be rem oved much earlier in the day. This will more accurately represent the day tim e status of levels of phosphor ylation in response to light in the complete absence of circadian efferent input. Figure 3-13: Preliminary (+)light to (-)li ght ratios determined by label free relative quantitation for each of three phosphoryl ation sites identifie d in the lateral eye. Error bars represent the 95% confidence interval for each phosphopeptide measurement determined from the relative 95% confidence interval of six non-modified control Lp MyoIII peptides. These data represent a single animal set, run in triplicate by HPLC-MS/MS. A second experimental modification will be made to test the effect of light on LpMyoIII phosphorylation. Previous studies show that a short-term exposure to light at midnight when the clock input to the eyes is active incr eases the concentration of LpMyoIII at the rhabdom. This change in LpMyoIII distribution may be due to light-

PAGE 95

81 driven changes in LpMyoIII phosphorylation, pe rhaps a change in the phosphorylation of the PKC site. We will test this by examin ing the phosphorylation levels of the four identified phosphorylation sites in LpMyoIII fo llowing removal of the eyes at midnight. Both eyes will have received normal clock inpu t, but one eye will be removed in the dark, while the other eye will be removed following a 20 min exposure to normal room light. Taken together, the two types of experime nts planned will provide clear data on the impact of light on LpMyoIII phosphorylation. Summary Four endogenous phosphorylation sites were verified in LpMyoIII by HPLCMS/MS, only three of which had been previ ously identified in recombinant LpMyoIII after in vitro PKA phosphorylation. A mass spect rometric method for the relative measurement of changes in levels of phosphor ylation at these site s was developed. The influence of the circadian clock on levels of phosphorylation at these sites was measured using this method, identifying two sites wh ich demonstrated increased levels of phosphorylation in response to clock input. Both of these are PKA sites; one of which has also been determined to be a substr ate for intermolecular auto-phosphorylation (Battelle et al. , 2004). The localization of these s ites to a region of myosins known to participate in actin binding, along with previ ous data, lend evidence to our hypothesis for a novel mechanism for regulating the actomyosin interaction based on local charge state modification. Preliminary data for the influe nce of light on phosphoryl ation at these sites has also been shown.

PAGE 96

82 CHAPTER 4 MASS SPECTROMETRIC EVALUATI ON OF 14-3-3 ISOFORM BINDING SELECTIVITY Introduction to the 14-3-3 Family of Proteins Phosphorylation-based signal transduction is typically viewed as a single step mechanism regulated by the presence and activ ation of specific kinases and phosphatases. The 1967 discovery of a class of acidic, solu ble, dimeric proteins of approximately 60 kDa in mammalian brain extracts (Moore a nd Perez, 1967) marked the first step in shifting this paradigm that would eventually prove highly ubiquit ous and significant. Named for their fraction in a chromatograp hic elution and subse quent position after starch gel electrophoresis (van Heusden a nd Paul, 2005), 14-3-3 pr oteins have been identified in every tested e ukaryotic organism (Ferl, 2004). Within these organisms, 143-3s are expressed in a wide ar ray of tissues and cell types. The 14-3-3 proteins serve as regulators of a second key step in ma ny phosphorylation-based signal transduction systems. In these systems, phosphorylation of the substrate results in no net change in substrate activity or signal propagation. In stead, substrate phosphoryl ation merely serves to increase that protei n’s affinity for 14-3-3 binding. It is only upon 14-3-3 binding to the phosphorylated substrate that the proverbial signal transduction “switch” is flipped. This added step of regulation represents some cellu lar need for more sophisticated control of the processes which employ 14-3-3s. To date, these include cell cycle control, apoptosis,

PAGE 97

83 transcriptional regulation, pr otein localization, protein tra fficking, and stress response (van Heusden and Paul, 2005). There are a number of ways in which the 14-3-3s manifest a response in the substrates they bind, and even more possible overall functions served by this family of proteins that are dependent on the substrat es themselves. One mechanism through which the 14-3-3s can exert effects on the substrat e involves a change in conformation, or stabilization of an otherwise transient confor mation, of the substrate, which results in an activation or deactivation of that substrate. Th is has been illustrated in the case of nitrate reductase, which is inactiva ted upon 14-3-3 binding (Huber et al. , 1996), and in the case of serotonin-N-acetyltransferase, which is fully activated by binding to 14-3-3 (Obsil et al. , 2001). Translocation of the substrat e following 14-3-3 binding has also been observed in a number of circumstances. For example, because 14-3-3s contain a conserved nuclear export signal, they can f unction to shuttle bound substrates out of the nucleus, such as is the case with the nucl ear export of the transc ription factor Cdc25 during interphase (Dalal et al. , 1999). The binding of 14-3-3s can also mask nuclear localization signals, blocking bi nding of nuclear shuttling pr oteins and inhibiting nuclear import (Sekimoto et al. , 2004, van Heusden and Paul, 2005). Due to the dimeric nature of the 14-3-3s, more than one binding site may exist per molecule, allowing for the stabilization or stimulation of protein comple xes or specific protei n-protein interactions (Braselmann and McCormick, 1995; van Heusde n and Paul, 2005). In addition, 14-3-3 activity can itself be regulated by post translational m odification, including phosphorylation (Aitken et al. , 1995), ubiquitination (Peng et al. , 2003), and acetylation (Martin et al. , 1993).

PAGE 98

84 Each 14-3-3 monomer is composed of nine antiparallel -helices that form a Ushaped groove when dimerized at the N-termini . While presently unresolved by X-ray crystallography, the C-termini are thought to compose the channel and/or flexible cap that regulates substrate accessibi lity to the binding groove (Truong et al. , 2002). Studies have indicated that divalent metal ion bindi ng at the junction of the C-terminus and the main structure regulates the moveme nt of this C-terminal tail (Lu et al. , 1994; Athwal et al. , 1998; Athwal and Huber, 2002; Sehnke et al. , 2006). Figure 4-1: Structural diagram of a 14-3-3 dimer. Each monomer is composed of 9 antiparallel helices, labeled 1-9. Helices 14 take part in forming the rigid hinge between the two monomers. Helix 9 com poses the flexible C-terminal tail. Divalent cation binding sites are located at the junction of helix 9 with the main structure (loop 8). The interior residues of each U-shaped monomer create a phosphopeptide bi nding groove. [Reprinted with permission from Sehnke, P.C.; DeLille, J.M.; Ferl, R.J. “Consumating Signal Transduction: The Role of 14-3-3 Proteins in the Co mpletion of Signal-Induced Transitions in Protein Activity.” The Plant Cell Supplement 2002 , S339-S354. Copyright 2002 by the American Society of Plant Biologists.] The 14-3-3s are represented by a relatively large multi-gene family with thirteen unique genes identified in Arabidopsis , seven in humans, and two in Drosophila . All of these genes encode for prot eins that demonstrate a hi ghly conserved core region,

PAGE 99

85 including the residues respons ible for binding to target phosphopeptide sequences (Liu et al. , 1995). A high degree of divergence is f ound in the extreme Nand C-termini, however, which form the junction of the dimer and the flexible cap over the binding groove, respectively. Increasing the complexity of this fami ly of proteins, 14-3-3s can exist as either homoor he tero-dimers. The added possi bility for post translational modification significantly increases the total number of active forms of 14-3-3 that may be present in a cell at a given time. The cellular purpose for such a large family of isoforms is not yet completely understood, though many studies are currently aimed at elucidating this purpose. Several general hypotheses exist, each of which may prove legitimate in a different context. One such perspective involves a random isoform divergence which would have occurred as a result of anatomical isolation and the evolutionary pr essure to preserve 143-3 function in different cell types and tissues. Biological control of 14-3-3 function in this case would occur at the level of mRNA and/or protein expre ssion patterns as opposed to preferential isoform bindi ng specificity. Support for th is hypothesis is found from multiple sources. First, isoform-specific expression in di fferent cell types and tissues has been observed in many organisms, indicating some degree of anatomical isolation (Testerink et al. , 1999; Daugherty et al. , 1996; Ferl, 2004). This was dem onstrated in rat brain, whose multiple 14-3-3 isoform mRNA levels were found to be differentially expressed in a manner dependent on neuron type and st age of cytodifferentiation (Watanabe et al. , 1994). The highly conserved nature of the di fferent isoforms, especially in the region responsible for substrate bi nding, also suggests a redundanc y in function and again a

PAGE 100

86 strictly random genetic evol ution of non-functionally signifi cant regions of the protein. Furthermore, multiple Arabidopsis 14-3-3 isoforms have been shown to resurrect normal function in yeasts that are de ficient in both of their 14-3-3 isoforms, an otherwise lethal mutation (van Heusden et al. , 1996; Kuromori and Yamamoto, 2000). Further emphasizing this concept, presumed knoc kouts of individual 14-3-3 isoforms in Arabidopsis failed to produce a notic eable phenotype (Krysan et al. , 1996). Another perspective dictates an evolu tionary divergence driven by functional requirement, yielding multiple isoforms that may exhibit more efficient interaction with preferred ligands. In this model, structural evolution would parall el the evolution of functional specificity and substrate specificity of the individual isoforms. Evidence in support of this hypothesis is slowly m ounting. A number of studies on the in vitro binding capabilities of various 14-3-3 isoforms with known s ubstrates such as nitrate reductase (NR) (Bachmann et al. , 1996) and H+ATPase (Rosenquist et al. , 2000) have demonstrated isoform specific binding preferences (Sehnke et al. , 2002). The use of isoform-specific antibodies has revealed s ub-cellular, as well as sub-organellar, organization of different isoforms (Sehnke et al. , 2000; Sehnke et al. , 2001), again suggesting isoform specific function and interaction. A 2005 study by Paul et al. demonstrated sub-cellular localization of sp ecific isoforms by the transformation of Arabidopsis plants with green fluorescence protein (GFP) fu sions of individual 14-3-3 isoforms. In addition, the differential spatia l distributions of the four evolutionarilydivergent isoforms studied were disrupted when treatments known to prevent normal 143-3 function or substrate inte raction were applied (Paul et al. , 2005). This result clearly

PAGE 101

87 suggests a substrate-driven lo calization of different isofor ms in the cell, supporting the hypothesis for a functional evolution of the 14-3-3 protein family. Regardless of the underlying purpose for this structural divers ity, an understanding of the factors intimately involved in subs trate binding would aid considerably in elucidating the factors responsib le for observed substr ate selectivity among the isoforms. Many studies have identified key amino acids in both the divergent termini and the mostly conserved binding groove whose subst itution greatly alters if not eradicates normal function of that isoform with regard to substrate binding. Whether the foundation for binding selectivity lies in the highly di vergent Cor N-termini, subtle amino acid substitutions within the cons erved core and binding groove, or a combination of the two is still not completely understood. Recent an alysis of exposed loop regions of 14-3-3s by Sehnke et al. confirmed loop 8 to be a critical re gion for divalent cation binding (Athwal and Huber, 2002; Lu et al. , 1994; Schultz et al. , 1998), and determined the influence of a single medial glycine in loop 8 (G213 in Omega and Nu) on C-terminal flexibility (Sehnke et al. , 2006). This medial glycine is conser ved in non-epsilon isoforms, but is substituted by a serine or arginine in ep silon isoforms, yielding an experimentally distinguishable reduction in C-terminal flexibility in the latter 14-3-3 sub-group. Mutation of this residue to a glycine in eps ilon isoforms was shown to render C-terminal flexibility similar to that of the non-epsil on isoforms according to proteolytic analysis (Sehnke et al. , 2006). This work stimulated interest in the dete rmination of whether this differential level of C-terminal flexibility between 14-3-3 sub-groups had any effect on target phosphopeptide binding. In this chapter, result s are presented from a mass spectrometric

PAGE 102

88 method, in conjunction with a 14-3-3 microaffinity capture technique developed previously by Sehnke et al. , which allow for the preliminary assessment of trends in phosphopeptide binding preferen ces of an epsilon, non-eps ilon, N213G-mutated epsilon, and a G213S-mutated non-epsilon isoform. Preliminary evidence for an effect on phosphopeptide binding preference as a result of the loop 8 po int mutation in the epsilon isoforms is demonstrated, while no apparent change is immediately noticeable as a result of the non-epsilon loop 8 mutation. These data indicate a possible ro le for this medial loop 8 site, and the C-terminally located loop 8 in general, in isoform-specific substrate selection, and justify further analysis involvi ng more stringent quantitative measures. Site-directed mutagenesis has also revealed a number of residues within or near the highly conserved binding pocket of 14-3-3s th at are critical to proper function. In particular, substitution/ mutation of importa nt sites within the hydrophilic face of the binding groove have demonstrat ed altered performance in all functional assays (Zhang et al. , 1997; Zhang et al. , 1999; Visconti et al. , 2003; Subramanian et al. , 2004). On the contrary, substitution mutations of importa nt sites within the hydrophobic face of the groove have shown significant changes in bind ing and activation of certain substrates (Visconti et al. , 2003; Subramanian et al. , 2004; Wang et al. , 1998), while having no consequence on other substrate systems (Zhang et al. , 1999). Furthermore, differential binding of phosphopeptides as well as to na tive phosphoproteins has been observed, a trend that would not be expected if subtle differences in the bi nding groove were not having some effect on substrate selection (Bachmann et al. , 1996; Roenquist et al. , 2000). To address the question of 14-3-3 selectivity in terms of the preferences defined by the mostly conserved binding groove, a ma ss spectrometric method was developed to

PAGE 103

89 study the relative binding affinities of eight synthesized mode-1 octamer phosphopeptides to two isoforms representing the two majo r evolutionary sub-groups, epsilon and nonepsilon. Normalized “binding ratios” were assigned to each phosphopeptide sequence with respect to each isoform such that co mparisons could be made in terms of the sequence preferences of each isoform as well as isoform-specific preferences for each sequence. Methods Protein Expression and Purification The pET15b 14-3-3 expression vectors were transformed into Escherichia coli BL21DE3 or BL21-AI (Invitrogen). Bacteria were grown in a 37 C shaking incubator to an optical density of 0.6 at 600 nm. Protei n expression was induced with addition of isopropylthio-galactoside (IPTG) to a final concen tration of 1 mM and bacteria were grown for an additional 2 hours, post-induction. Bacteria were pelleted by centrifugation twice at 5000 x g for 7 minutes each, and bacteria l pellets were collected and put on ice. Lysis buffer (50 mM Tris, 2 mM MgCl2, 20 mM NaCl, 0.2% Triton-X-100, pH 8.4, with protease inhibitors and 1:1000 lysosyme) wa s added, incubated on ice for 20 min, and frozen at -80 C. Upon thawing, 1 l/ml benzonase was added and the mixture was again kept on ice until solution became clear (indi cating DNA and RNA had been degraded). Proteins were pelleted by centrif ugation at 10,000rpm for 20 minutes at 4 C, twice. Final protein pellets were resuspended in 10 ml of 50 mM Tris, 300 mM NaCl, 10 mM imidazole, and 0.2% Triton-X-100 (pH 8.0). Hi s-tagged 14-3-3s were purified using the Ni-NTA HisBind purification k it (Novagen). Purified 14-3-3s were dialyzed into phosphate buffered saline (9.1 mM diba sic sodium phosphate, 1.7 mM monobasic

PAGE 104

90 sodium phosphate, 150 mM NaCl; pH 7.4) overnight at 4 C. Final protein concentrations were determined by spectromet ric absorbance at 280 nm. Phosphopeptide Synthesis Phosphopeptides were synthesized by the pe ptide synthesis group at the University of Florida’s ICBR Proteomics Core by solid phase FMOC-chemistry using an Applied Biosystems (Foster City, CA) peptide s ynthesizer (model 432A). Eight phosphopeptide octamer peptides were synthesized based upon the nitrate reductase (NR) mode-1 14-3-3 binding sequence, KKSVpSTPF. These seque nces represented four p+1 (denoting location relative to th e phosphorylated serine) and four p-1 modifications. Specifically, these sequences were: KKSVpS Y PF (NR p+1 “Y”) KKSVpS I PF (NR p+1 “I”) KKSVpS A PF (NR p+1 “A”) KKSVpS M PF (NR p+1 “M”) KKS Y pSTPF (NR p-1 “Y”) KKS F pSTPF (NR p-1 “F”) KKS S pSTPF (NR p-1 “S”) KKS T pSTPF (NR p-1 “T”) (modified residues shown in bold). The NR binding sequence (referre d to as wild type, WT, in following text) was also synthesized in its natural “light” (WT) and stable-isotope labeled “heavy” (WT*) form. Synthesis of WT* was accomplished via substitution of the unlabeled C-terminal phenylalanine with L-phenylalanine-N-FMOC (13C5, 15N1) (Cambridge Isotope Laboratories, Inc., Andover, MA). Buffer Optimization for ESI-MS Coupled Microaffinity Chromatography A stock solution of 100mM morpholinoeth anesulfonic acid (MES) was prepared and serial dilutions were made to yield fi ve test concentrations: 40 mM, 4 mM, 0.4 mM, 0.04 mM, and 0.004 mM. Each test solution was made in 50% methanol, 1% acetic acid,

PAGE 105

91 and each contained 2.8 M NR p+1 “A” and 2.8 M NR p+1 “M”. Each test solution was analyzed by direct infusion ESI-FTICRMS in triplicate, and phosphopeptide ion signal intensity was measured. Averaged phosphopeptide signal intensities for each of the test solutions were plotted agains t their corresponding MES concentrations. A stock solution of 50 mM ammonium bicarbonate was prepared and serial dilutions were made to yield five test concentrations: 10mM, 1.0 mM, 0.1 mM, 0.01 mM, 0.001 mM. Each test solution was made in 50% methanol, 1% acetic acid, and each containing 2.8 M NR p+1 “A” and 2.8 M NR p+1 “M”. A second set of these solutions was made with 0.1% acetic acid. E ach test solution was analyzed by direct infusion ESI-FTICR-MS in triplicate, a nd phosphopeptide ion signal intensity was measured. Averaged phosphopeptide signal intens ities for each of the test solutions were plotted against their corresponding amm onium bicarbonate concentrations. 14-3-3 Microaffinity Capture Chromatography Millipore ZipTip MC (one per experimen t/isoform) were aspirated with 400 mM NiSO4 at least 5 times in order to charge the metal chelating column with Ni2+. The charged columns were washed 4 time s with 10mM Tris, 250mM NaCl, 2.5 mM imidazole (buffer pH 7.9), which will be referr ed to as buffer A in the following text. His-tagged, recombinant 14-3-3 isoforms were diluted to a final volume of 30 l in buffer A to achieve a final protein c oncentration of approximately 0.1 g/ l. Protein solutions were aspirated slowly through the ZipTips a pproximately 5 times and tips were allowed to incubate in the remaining solution for at least 15 minutes. After incubation, 14-3-3 affinity ZipTips were washed four time s with buffer A to remove unbound protein. Approximately 10 g of the mixture of synthetic phos phopeptides, to which the synthetic

PAGE 106

92 WT peptide had been added at a 1:1 ratio (for quantitative experiment s), were diluted to a concentration of approximately 1 g/ l in 20 mM HEPES, 300 mM NaCl, 5 mM MgCl2 at pH 7.2 (buffer B from this point forwar d). This phosphopeptide solution was slowly aspirated through the 14-3-3 microaffinity co lumn at least 5 times, and the tip was incubated in the phosphopeptide solution fo r 30 minutes. Phosphopeptide bound affinity columns were washed rapidly with two 20 l volumes of buffer B, followed immediately by three 20ul volumes of 10mM ammonium bi carbonate (pH 6.8) in order to completely remove unbound phosphopeptides. 14-3-3-boun d phosphopeptides were eluted by at least five aspirations of the ZipTip with 10 l of 1% acetic acid. Either 5 l of 100% methanol (phosphopeptide profiling experiments) or 5 l of 475 nM WT*, 1.0% acetic acid in methanol (for quantitative experi ments) was added to the final eluents in preparation for mass spectrometric analysis. Mass Spectrometric Instrumentation and Operational Parameters A Harvard Apparatus (Holliston, MS) syringe pump was used to deliver samples at a flow rate of 0.5 l/min to the Agilent (Waldbronn, Germany) off axis ESI source configured with nitrogen nebulizing gas. A Bruker Daltonics (Billerica, MA) BioApex II 4.7 Tesla FTICR-MS was used for all analyses. Electrospray voltages were applied in the approximate range of -2800 to -3400 V to th e end cap electrode, creating a potential between the grounded electrospray needle and the entr ance to the source region sufficient to initiate and maintain spray. All sour ce parameters (end plate, capillary entrance, capillary exit, skimmer 1, skimmer 2, hexa pole DC offset, trap, extract, hexapole accumulation time [D1], and hexapole to cell f light time [P2]) were optimized prior to each analysis (at least once a day). Ion optic s (series of focusing cylinders and deflection

PAGE 107

93 plates) were optimized only once every 1 – 3 weeks based on sample load. Mass calibration was performed daily with HP T uning Mix (Agilent Technologies, Palo Alto, CA). Calibration was facilitated by a 3 or 4 point non-linear calibration algorithm provided by Bruker’s Xmass software. Ions were captured in the cell by SideKick trapping and data collected in broadband m ode. Data were typically collected in 1 Mpoint files (indicating length of transient collected), 4-100 scans were added (based on initial signal intensity), and final spectra were apodized and transfor med. Peak lists with absolute ion intensities of all peaks within the mass range of interest were generated using Xmass. The m/z and absolute ion inte nsity values were used for any subsequent quantitative analysis. Results Optimization of Affinity Capture Exp eriment for Mass Spectrometric Coupling The 14-3-3 affinity capture experiment ha d been created and optimized for optimal phosphopeptide binding prior to coupling with di rect infusion ESI-MS. In the original procedure, (MES) was required in the phosphope ptide binding and wash buffers in order to control pH for preservation of 14-3-3 isoform activity and bi nding capability. Though no MES was present in the elution buffer, a single column volume (~0.5 l) of the wash buffer containing 4mM MES was eluted with the peptides into the final solution. Calculation of the final con centration of 0.06 mM MES in the solution to be used for direct infusion ESI-MS (Fig. 4-2 A) generate d concern with regard to ESI efficiency, since this concentration approaches th e maximum threshold concentration (10-5 M) for analytes in ESI (Enke, 1997). The effect s of increasing MES concentration were analyzed via direct infusion ESI-FTICR-MS under otherwise identical sample conditions (approximate peptide concentrati on, instrumental parameters). It was determined that

PAGE 108

94 Figure 4-2: Effect of MES on electrospra y ionization efficiency of phosphopeptides. A) Calculation of final concentration of MES in spray buffer, assuming a 0.5ul column volume (amount of wash buffer left on column after final wash) and considering subsequent dilutions. B) Measurement of the absolute intensity of phosphopeptides present at equal c oncentrations, but with increased concentration of MES buffer in solu tion. A rapid decline in phosphopeptide ESI efficiency is apparent as buffer con centration is increased. A greater than 50% decrease in signal intensity is observed at the approximate MES concentration calculated to remain in the final 14-3-3 microaffinity ZipTip eluate. Error bars repres ent 1 standard deviation. ESI signal was decreased more than 50% at a concentration of 0.04 mM MES, which corresponds to the approximate calculated con centration remaining in the sample after

PAGE 109

95 elution. ESI signal continued to show a ra pid decline with each order of magnitude increase in MES concentration (Fig. 4-2 B). As a result, an alternative volatile buffer which maintains a pH similar to that of MES, ammonium bicarbonate, was tested for compatibility with ESI. No effect on electrospray efficiency was observed as the concentration of amm onium bicarbonate in the solvent system was increased (Fig. 4-3), va lidating the use of this buffer in these ESIcoupled experiments. Figure 4-3: Effects of amm onium bicarbonate concentratio n on electrospray ionization efficiency of synthetic phosphopeptides. No decline in ESI efficiency was observed with an increase in ammoni um bicarbonate conc entration in the analyte solution, justifying use of this buffer in subsequent affinity binding experiments. Error bars re present 1 standard deviation. Negative control experiments were conducte d to ensure complete removal of nonspecifically bound synthetic phosphopeptides from the metal chelating resin in the microaffinity experiments. Experiments carr ied out on ZipTips that had not been charged with Ni2+ produced no evidence of non-specifi cally bound phosphopeptides. Conversely, in experiments where the resin had been charged with Ni2+, but either not bound with any protein or bound with a Hi s-tagged non-phosphopeptide bind ing protein ((-) control

PAGE 110

96 protein in Fig. 4-4), some non-specific bi nding of the synthetic phosphopeptide mixture was detected. To correct for this effect , the number of salt/buffer washes postphosphopeptide incubation was increased from 3 to 5, which seemed to elliminate detectable non-specific binding to the Ni2+-charged resin (Fig. 4-4). Figure 4-4: Determination of number of salt washes necessary post-phosphopeptide incubation to eliminate non-specific binding of phosphopeptides to the Ni2+ charged resin. In the left panel is a representative spectrum demonstrating non-specific binding of two phosphopeptides to the Ni2+ charged resin with a bound negative control protein when only 3 salt washes are performed after phosphopeptide incubation. After 5 salt washes, detectable non-specific binding is eliminated (right panel). Effects of Loop 8 Point Mutation on Phosphopeptide Binding The effects of single point mutation at a critical amino acid in loop 8 on phosphopeptide binding were surveyed by 14-33 microaffinity capture chromatography in conjunction with mass spectrometry. Pr ior to microaffinity chromatography, the phosphopeptide mixture was first analyzed by di rect infusion ESI-FTICR-MS in order to visualize the unaltered phosphopeptide profile. Figure 4-5 demonstrates a representative “pre-binding” phosphopeptide prof ile, which was found to be highly reproducible in our analyses. Differences in relative ion intensit ies in this spectrum were a reflection of peptide synthesis efficiencies, mixing effici encies, ionization efficiencies, and matrix effects on ionization.

PAGE 111

97 Figure 4-5: Representative direct infusion ESI-FTICR-MS spectrum of the “before binding” phosphopeptide mixture. Peaks are annotated with the location and type of modification to the native NR binding sequence. Relative peak intensities in this spectrum reflect synthesis efficiency, mixing efficiency, ionization efficiency, and matrix effects on ionization for each phosphopeptide relative to othe rs in this mixture. In order to determine the effects of point mutation on binding at a sub-group level, the ‘after-binding’ profiles of a non-epsilon and epsilon isof orm were compared to their mutated counterparts. Figure 4-6 shows the pos t binding spectra of Nu (non-epsilon) and Nu G213S, along with Mu (epsilon) and Mu N213G for MS profile comparison. For the Nu isoforms, no stark deviation for the general post binding profile wa s witnessed in the mutated Nu isoforms. While minor shifts in relative peak intensi ties were observed, the significance of this needs to be validated by additional analyses incorporating internal standards for phosphopeptide peak intensit y comparison. The Mu N213G spectra, however, revealed stark differences from the non-mutated Mu profile, indicating a possible alteration of phosphopeptid e selection and binding affin ity as a result of single

PAGE 112

98 point mutation at this medial loop 8 residue. Again, repeat ed analyses with internal standards would be required to fully char acterize the effects of N/S213G mutation on phosphopeptide selection for this sub-group of 14-3-3 isoforms. Figure 4-6: ‘After-binding’ phosphopeptide profiles of a non -epsilon (A) and epsilon (C) isoform, along with their G/N213-mutate d counterparts in panels B and D, respectively. No drastic change in the MS profile is observed between the non-mutated and mutated Nu isoform, indicating little to no change in selectivity of binding as a result of the G213S mutati on. In panels C and D, the phosphopeptide labeled in red highlight s the most apparent change in the ‘after-binding’ phosphopeptid e MS-profiles be tween the non-mutated Mu and Mu N213G isoforms. This result provid es preliminary evidence for a change in the target selectivity of epsilon isof orms as a result of the N213G mutation. These experiments clearly present the need for further development of an analytical method which would allow direct comparis on of phosphopeptide peaks both within a single ‘after-binding’ spectrum and between ‘after-binding’ sp ectra of different isoforms or mutated isoforms. This method should also take into account the effects of differential efficiency in peptide synthesis, mixing, i onization, and matrix effect which can be

PAGE 113

99 measured as a relative total effect in the “before-binding” spectr a of the phosphopeptide mixture. An understanding of the sub-gr oup specific (or non-sp ecific) phosphopeptide sequence preferences would also aid in profile analysis of mutated isoforms, as well as provide valuable information toward understa nding the local sequenc e preferences of 143-3s in their target interactions. Analysis of 14-3-3 Sub-Group Specific Target Phosphopeptide Sequence Preferences In order to normalize a phosphopeptide ion’ s absolute intensity in a mass spectrum such that this value would represent the affi nity of that phosphopep tide toward the 14-3-3 isoform in question, internal standards were introduced and a coll ective linear dynamic range (LDR) and lower limit of quantitation (LLOQ) were established. Two internal standards were chosen for these analyses: the nitrate reductase 14-3-3 binding peptide (WT), and a stable isotope labeled WT (WT* ) with a 6 amu mass sh ift (see methods). The WT control peptide was spiked into th e phosphopeptide mixture pr ior to the binding experiment, and served as the normalizing f actor for each analyzed phosphopeptide. The WT* control peptide was spiked into the mixture after the binding experiment, and served as a determinant of analysis within the determined linear dynamic range. Figure 4-7 is a schematic diagram of the experimental work-flow. Various molar quantities of both the WT a nd WT* control peptides were examined such that final ‘after-bindi ng’ spectra yielded approximate ly 1:1 ratios of the average phosphopeptide signal intensity to the signal intensities of each of the control peptides. It was determined experimentally that the WT pe ptide should be spiked into the pre-binding phosphopeptide mixture at a 1:1 molar ratio (m oles WT to approximate moles of each phosphopeptide) in order to yi eld an approximate 1:1 signa l ratio between the average phosphopeptide signal and WT signal in the af ter binding spectra (d ata not shown). A

PAGE 114

100 final concentration of 158 nM WT* was found to typically yield an approximately 1:1 signal ratio with the average phosphopeptide signals in the af ter binding mixture. As a result, 5 l of 475 nM WT* in methanol was added to each 14-3-3 microaffinity column effluent (instead of 5 l of 100% methanol added for prof iling experiments) in order to achieve this concentration in the final sample solutions. Figure 4-7: Schematic diagra m of experimental work-flow for quantitation of relative binding affinities of phosphopeptides to specific 14-3-3 isoforms. WT peptides are spiked into phosphopeptid e mixture at an approximately 1:1 molar ratio prior to binding. This internal standard will serve as the normalizing factor for all phosphopeptides. After the binding experiment is completed, WT* is spiked into the eluate in methanol to yield a final concentration of 158 nM. This internal standard will be used to ensure analysis within the linear dynamic range of the method. The final mixture is analyzed by direct infusion ESI-FTICR-MS. Standard curves were constructed in order to determine a linear dynamic range (LDR) and lower limit of quantitation (LLOQ). Plots of both the approximate phosphopeptide concentration to measured phosp hopeptide/WT ratios (Fig. 4-8) and each approximate phosphopeptide concentration to measured phosphopeptide/ WT* ratio (Fig. 4-9) provided analogous results. A collec tive LLOQ for these ratio measurements was

PAGE 115

101 determined to be ~100 nM for all phosphopep tides (Fig. 4-10), si nce linearity of the standard curves was generally lost below th is concentration. Anal yses remained linear over an order of magnitude above this concen tration. Concentrations over one order of magnitude greater than the LLOQ (our upper LDR limit tested) were not analyzed, since all ‘after-binding’ phosphopeptid e concentrations were determined to be well below the upper LDR limit tested (~ 1 M). Figure 4-8: Plot of a pproximate phosphopeptide concen tration vs. phosphopeptide/WT signal intensity ratio. WT is spiked into all standard solutions at an ~1:1 ratio. All plots should have a theoretical sl ope of zero within the linear dynamic range of the method. Near-zero slopes are demonstrated for all peptides over an order of magnitude in the practical range of sampling, justifying use of this method over the investigated linear dyna mic range. Error bars represent the 95% confidence interval for all measurements determined from three analyses at each data point.

PAGE 116

102 Figure 4-9: Plot of approximate phosphope ptide concentration Vs. each phosphopeptide/ WT* signal intensity ratio. All plots demonstrate positive slopes in their linear dynamic range which reflect each phosphopeptide’s response factor relative to WT*. Equations of these lin es will be used in determination of analysis within the linear dynamic ra nge of each analysis. Error bars represent the 95% confidence interval of all measurements determined from three analyses per data point. Figure 4-10: Determination of LLOQ. Zooming in on the lower concentration range of standard curves reveals a loss in linearity for all phosphopeptides at a concentration of approximately 100 nM. This defines the collective LLOQ for all analyses. This value can be converted to a minimum phosphopeptide to WT ratio by insertion into the equations defined in Fig. 4-9. All experimental values found to be less than these minimum ratios were excluded from the final analysis.

PAGE 117

103 Using the established LLOQ value, a lo wer phosphopeptide/WT* ratio could be determined using the equations defining each tr end line in the response factor standard curve (Fig. 4-9). In all following quanti tative binding analyses, ‘after-binding’ phosphopeptide/WT* ratios were compared to th is lower ratio limit. All experiments yielding phosphopeptide/WT* ratios below this minimum value were excluded from final calculations. With these values established, the bind ing experiments described were conducted eight times on both 14-3-3 omega (non-epsil on isoform) and 14-3-3 epsilon (epsilon isoform). Each phosphopeptide ion’s absolute intensity in the ‘after -binding’ spectra was normalized to the WT ion’s absolute intensity from the same ‘after-binding’ spectra. These normalized signal intensity values were themselves normalized by the respective phosphopeptide/WT ratios determined from the before binding spectra (equation 4-1). Equation 4-1 : This final normalization step removes effect s of differential peptide synthesis, mixing, ionization efficiency, and susceptibility to ma trix effects, such that the final reported value provides a relative meas ure of each phosphopeptide’s bi nding affinity to the 14-3-3 isoform in question. The results of these experiments are su mmarized graphically in Figure 4-11. The two isoforms, representing the two main subs ets of 14-3-3 isoforms, demonstrate similar phosphopeptide selectivity. Only the NR p+1 “A” peptide displayed unique binding affinity between the two isoforms, however fo r both isoforms this peptide was found to

PAGE 118

104 Figure 4-11: Relative measure of each phosphopeptide’s binding affinity to each isoform, normalized against the NR WT peptide. Eight experiments were averaged for each peptide, unless otherw ise noted. Error bars represent the 95% confidence interval for each measurement. Table 4-1: Average “before binding” phos phopeptide/ WT ratios, average “after binding” phosphopeptide/WT ratios, their associated standard deviations, and final “binding ratios” with propagated error analysis.

PAGE 119

105 be the strongest binder. NR p-1 “T”, a low intensity peptide in ‘b efore-binding’ spectra, was never detected in ‘after-binding’ spect ra, and therefore no inference on this phosphopeptide’s binding affinity could be made. Discussion A preliminary survey of the selectivity in phosphopeptide binding for a non-epsilon 14-3-3 isoform, an epsilon 14-3-3 isoform, and mutated versions of each of these isoforms was conducted. Previous studies by the Ferl lab (Sehnke et al. , 2006) had established the importance of a medial residue in loop 8 which played a profound role in determining C-terminal flexibility. Conser ved in non-epsilon isof orms as a glycine residue, and in epsilon isoforms as either a serine or aspa rigine residue, observed Cterminal flexibility correlated di rectly with the bulk of the si de chain at this medial loop 8 position. This residue’s influence on flexib ility was confirmed by single point mutation of the loop 8 medial asparagine to glycine in an epsilon isoform, followed by proteolytic analysis (Sehnke et al. , 2006). Because of the observed ch anges in C-terminal flexibility, a region of 14-3-3s expected to play a major regulatory role in substrate selectivity, an assay for profiling binding pref erences of mutated isoforms relative to their non-mutated counterparts was developed. It was determined through this preliminary work that single point mutations at the medial loop 8 residue may alter phosphopeptide selectivit y in epsilon isoforms, since differences in the non-mutated and muta ted epsilon isoform ‘after-binding’ phosphopeptide profiles were observed. In contrast, only subtle and probably insignificant changes in the ‘after-binding’ phosphopeptide profile were apparent in mutated non-epsilon isoforms as compared to their non-mutated counterparts. These data justify further analysis of these trends. A characterization of clear and reproducible

PAGE 120

106 changes in the phosphopeptide selectivity as a re sult of the single point mutation in loop 8 or C-terminal truncation would provide substa ntial evidence for the crucial role of the highly divergent C-terminus in isoform-speci fic substrate selectivity. To draw such conclusions for the MS-profiling experiments described here, a more sophisticated means of measuring changes in leve ls of binding was required. As a result, a mass spectrometric method for the quantitation of relative binding affinities (with arbitrary units) of eight s ynthesized phosphopeptide sequences to different 14-3-3 isoforms was developed. This method’ s capabilities were demonstrated using two isoforms that represented the two main sub-gr oups of the plant 14-3-3 family, and can be applied to the analysis of a ny 14-3-3 isoform or mutant. Fi nal values obtained from this method can be compared in any combination; i.e. to interpret different sequence preferences of a single is oform, or to determine differences in selectivity of a particular phosphopeptide sequence between two or more isoforms. Using the phosphopeptide sequences chosen for this study, no isoform-specific substrate selectivity was obser ved between omega (non-epsilon) and epsilon (epsilon). However, reproducible trends in sequence pr eference consistent be tween both isoforms were apparent. For the p+1 modified phosphopep tides, a clear preferen ce for the smaller, aliphatic, hydrophobic alanine over the naturally occurring threonine in this position was observed, with relative binding affinities ~10 ti mes and ~25 times greater than that of WT for non-epsilon and epsilon isoforms, resp ectively. Two other phosphopeptides with aliphatic, hydrophobic amino acids in this position, however, demonstrated binding affinities similar to the WT sequence, making it difficult to presume whether the alanine substitution represents a trend in side chain chemistry sele ctivity. Substitution of the

PAGE 121

107 threonine with aromatic tyrosine in this pos ition resulted in a three fold increase in binding affinity for both isoforms (Fig. 4-11). Substitution in the p-1 position resulted in a seemingly simpler set of trends in side chain chemistry preference. The nitrate re ductase sequence, after which all synthetic phosphopeptides were modeled, bears an al iphatic, non-polar, hydrophobi c valine at this position. Substitution with the polar, hydrophilic se rine resulted in a subtle decrease in binding affinity of the phosphopeptide to both isoforms. On the other hand, replacement of valine with either a phenylalanine or a tyrosine, both aromatic amino acids, demonstrated similar degrees of approximate ly 3 fold increased binding affinity. Future Directions Continued progress on this project may proceed along two parallel courses. Biologically, a new set of questions has now been posed. In terms of 14-3-3 substrate selectivity as a result of structural featur es localized to the mo stly conserved binding groove: Are the observed trends in the posit ions of particular amino acid side chain chemistries relative to the phosphorylated serine in the 14-3-3 binding sequence consistent among other isoforms? How flexible is the requirement for certain chemistries in these positions? What type s of interactions are governing the selection of these amino acids? Does modification of amino acid side chain chemistry at any position near the phosphorylated serine result in is oform specific selectivity? In terms of the effects of the C-terminus and the medial loop 8 residue on 14-3-3 binding: Utilizing the quantitative method developed, would the differences in binding efficiency be tween mutated and nonmutated isoforms observed here be confirmed? If so, complete characterization of these differences in epsilon and nonepsilon isoforms would be a dvantageous. Do C-terminal truncations to the 14-3-3s alter phosp hopeptide binding? If isoform-specific

PAGE 122

108 phosphopeptide selectivity is found, does truncati on of the C-terminus eliminate or alter this selectivity? In terms of the mass spectrometric method developed, pursuing alternative, more automated instrument platforms would facilitate analysis of large sample sets. While the FTICR-MS was used in these studies due to its high resolving power and ability to accurately detect and provide a measure for all phosphopeptides in a small mass range simultaneously, mass spectrometers with less re solving power could be utilized for this study when conjugated with an additional separation technique such as HPLC. For example, the Thermo LCQ Deca 3D ion trap HPLC-MS/MS system is capable if separating phosphopeptides from a mixture in time by reversed-phase HPLC directly coupled to the ion trap MS. The mass range scanned by the ion trap can be confined in the instrument method to the short range necessary for analysis of the phosphopeptide mixture. Reducing scan speed over this shorter mass range can result in increased resolution. The LC time frame may also be segmented in the instrument method, such that the instrument can target for specific clusters of phos phopeptides eluting at similar times during different segments of the LC time frame (Fig. 4-12). The ability to assign particular scan events to the fragmentation of a specific peptide increases the sensitivity of analysis. Isolation and di ssociation of a defined and na rrow mass range regardless of detection of the parent ion in the full scan can often result in the iden tification of daughter ions in the MS/MS spectra characteristic of the precursor ion of interest. This is especially true in phosphopeptide analysis, since resonant excita tion in the ion trap typically leads to neutral loss of the phospha te group, yielding a predominant ion in the MS/MS spectra which corresponds to the parent ion minus the phosphate group.

PAGE 123

109 Thermo’s XCalibur software enables the au tomated integration of the extracted ion chromatogram produced based on the presence of the correct neutral loss peptide ion in MS/MS spectra from the appropriate LC segment. Figure 4-12: Demonstration of phosphopeptid e separation by reve rsed phase capillary HPLC and subsequent ion trap MS detection. The top panel shows a representative base peak chromatogr am, indicating the elution times of different peak clusters, denoted A-F. A representative mass spectrum from each major peak in the base peak chromatogram is shown in panels A-F (corresponding to the appropriate peak in the base peak chromatogram) and the phosphopeptide(s) detected at each time point are labeled.

PAGE 124

110 Using the Thermo LCQ Deca ion trap for these analyses, samples could be prepared and data processed as described previo usly in this chapter, using the integrated peak areas instead of absolute peak intensitie s. The automated nature of this system, as well as its robust instrument parameters (t uning required at-most monthly as opposed to at-least daily) will greatly improve reproducibil ity of analyses as well as ease of data collection. Summary Preliminary evidence for a change in phosphopeptide binding se lectivity following mutation of an epsilon isoform at a key me dial residue within loop 8 was shown. The data collected in these experiments justif ied the development of a more stringent quantitative method to measure differences in the selectivity of is oform binding. Such a method was developed. Using this method, the degree to which a given phosphopeptide binds to a given isoform can be assigned a re lative value, defined here as the “binding ratio.” The binding ratio of a phosphopeptide w ith respect to a certain isoform can be directly compared to binding ratios from ot her phosphopeptides with respect to the same isoform, or to its own binding ratios with respect to differe nt isoforms. This system provides a simple measure of relative bindi ng affinity and a means for visualizing differences of changes in these binding affi nities. This method was applied to two isoforms representing the two main sub-gr oups of the 14-3-3 family, non-epsilon and epsilon. A reproducible preference for certa in phosphopeptide sequences was measured for both isoforms. This preference proved to be consistent among the two isoforms. These studies demonstrate the util ity of this method and justify its use in future analyses.

PAGE 125

111 CHAPTER 5 CONCLUDING REMARKS The work described in this dissertation follows a general theme, that of the mass spectrometric measurement of biological change . Mass spectrometry has proven itself a valuable tool in terms of its impressive abil ities to target, detect, resolve, and quantitate an analyte of interest. Advancements in mass spectrometric technology continue to keep mass spectrometric methods on the forefront of biological research, as their capabilities are improved with applications in mind. The growing reputation of mass spectrometry as a powerful tool in biologi cal research has been met, however, with unreasonable expectations. The proverb which has become familiar among those in the field of biological mass spectrometry, “garbage i n, garbage out,” succi nctly highlights the necessity for careful sample preparation techniques. While mass spectrometry can provide an excellent means of detection and measurement, the care taken in experimental planning and design (especially for steps that precede mass spectrometric analysis) will determine the success of these experiments and the scope of the information that can be drawn from these analyses. In general, the greater the inve stment in sample preparation, the greater the mass spectrometric return. Equally important in maximizing the amount of useful information drawn from mass spectrometric analyses is the focus plac ed on data processing and interpretation. The need for sophisticated software for interpretation of the overwhelming amounts of data that are collected with each ‘data-depende nt’ or ‘information-dependent’ analysis is being answered by the bioinformatics comm unity. While these algorithms greatly

PAGE 126

112 facilitate data analysis and enable the conversion of large amounts of ‘data’ into large amounts of ‘information,’ it is our responsibil ity to validate the au thenticity of this ‘information.’ Manual validation of software-g enerated results shou ld be considered a rule in data interpretation. The ability to produce so much ‘information’ can also misguide our efforts in terms of experimental planning. In general, a rift between ‘what was found’ and ‘what it means’ seems to be slowly growing in the field. Focus is often placed more on producing more information than on in terpreting the information. It is easy to consider advancement in the field of biological ma ss spectrometry as a game of numbers as instrumental capabilities and data processing software beco me more robust. Providing long lists of information certainly has its place in biological research, as these “lists” have become the ‘microarray of proteomics’ and a springboard for new research. In general, however, this philosophy has ge nerated frustration within the biological community whose questions are more specific and directed. Many la bs are beginning to consider this rift and to incorporate additiona l experiments into a research plan such that the question of ‘what it means’ can be addressed. In addition to added analyses, a preliminary reflection on what information is sought biologically c ould dictate different instrument or sample preparation methods that may produce less overall data, but more valuable biological information. This is also true of data processing techniques. By keeping attention on ‘what it could mean’ from the initial steps of experimental planning, we can continue to close this rift by fine tuning sample and data processing methods to each application. Ultimately, it is the accumu lation of specific and reproducible answers to specific biological questions that fuels the progression of research in this field and

PAGE 127

113 introduces new questions to be answered. In order to bridge any rift between ‘what was found’ and ‘what it means’ that may be left open after reading the preceding chapters, a brief look into ‘what it could mean’ in the broa der context will be e xplored here, and the niche to which each project belongs within its respective biological field will be identified. Class III Myosins: From Horseshoe Crabs to Humans The class III myosins are being studied in many organisms in order to determine the possible functions of these proteins. As the most divergent member of the Myosin superfamily, prediction of possible function fo r this class based on sequence similarities with other classes of myosins is complicated. The clear presence of an N-terminal kinase domain, a feature unique to the class III myos ins, indicates its likely role as a signaling molecule in all organisms. On the othe r hand, actin-activated ATPase activity and resultant motor function appear to vary among class III myosins from different organisms (Komaba et al. , 2003; Kempler et al. , submitted). Class III myosins have been found to be expressed predominantly in photoreceptors in most organisms; however in humans, occu rrence in kidney, testes , and ears in addition to the retina has been observed (Dos and Burnside, 2002; Walsh et al. , 2002). In fact, Dos and Burnside identified two genes c oding for human myosin IIIs (Myo3A and Myo3B) (Dos and Burnside, 2000; Dos and Burnside, 2002). Walsh et al. found that non-mutated, functiona l Myo3A is required for normal hearing in humans (Walsh et al. , 2002). Three specific genetic mutations in Myo3A were identified to result in inherited progressi ve nonsyndromic hearing loss. Myosin IIIA represents the fifth myosin found to be associ ated with human heari ng loss as a result of

PAGE 128

114 natural mutations. Mutations in Drosophila myosin III lead to retinal degeneration (Montell and Rubin, 1988); however, as of yet mutations in vertebrate myosin III have not been associated with abnormal retinal f unctions. This may be a result of myosin III redundancy in the retina; both Myo3A and Myo3B are present. Alte rnatively, different genomic mutations or disturbances to signaling cascades involving myosin III at the protein level may lead to human retinal dysfunction. Understanding how myosin IIIs contribute to pathologies is hindered by the limited information available on the functions of myosin III under normal physiological conditions. It is therefore im portant to elucidate the bioche mical properties of myosin IIIs, as well as their roles in cellular processes. Recently, a study by Komaba et al. (2003) demonstrated intra-molecular autophosphorylation of human myosin IIIA. Th eir evidence, based on biochemical and molecular experiments, suggested human myos in IIIA autophosphorylates at two sites, on a threonine in the kinase domain and a seri ne located within a 20kDa peptide in the Cterminal end of the motor domain (Komaba et al. , 2003). This 20kDa peptide includes a portion of loop 2, the actin-binding region of myosins which has been the focus of much of the work described in chapter 3 of this dissertation. The exact sites of phosphorylation in the human myosin III have not ye t been identified, however. Komaba et al. ( 2003) propose that autophosphorylation within the kinase domain e nhances myosin III kinase activity, however no other functional conseque nces were assigned to the phosphorylation within the C-terminus of the myosin doma in. It was shown in chapter 3 of this dissertation that LpMyoIII is also phosphoryl ated near the C-terminal region of the myosin domain and the location of one of these sites within loop 2 was positively

PAGE 129

115 identified. Previous studies using recombinant LpMyoIII demonstrated autophosphorylation of this l oop2 site as well (Battelle et al. , 2004). It may be that second messenger and/or autophosphorylation of class III myosins within or near loop2 will prove a common feature of class III myos ins across species, including humans, and the phosphorylation in this region may be a key to understanding the function of the protein in different species. The studies described in Chapter 3 are th e first to demonstrate the phosphorylation of sites within loop 2 in any myosin. No tably, this phosphorylati on event was identified in the endogenous protein following its in vivo response to a natural stimulus. Identifying the specific phosphorylation site s in the myosin domain of LpMyoIII that change their level of phosphorylation in res ponse to specific stim uli, in this case circadian clock input (and preliminary results describing the effect s of light) points to possible functions of myosin III in Limulus photoreceptors, and identifies a possibly novel mechanism for acto-myosin interaction (see Chapter 3 discus sion). Comparison of results from this work with the results of studies on human myosin III by Ikebe et al. , (2003) reveal possible structural and functiona l similarities between the tw o myosin IIIs, and provide a framework for future studies on human myosin IIIs and their relation to associated diseases provided. The 14-3-3 Protein Family: From Common Weeds to Humans The family of 14-3-3 proteins studi ed in chapter 4 was derived from Arabidopsis and represented the plant family of 14-3-3s. This family includes 13 isoforms whose functional specificities have not yet been comp letely elucidated. The work in chapter 4 focused on identifying general trends in binding selectivity am ong the two major subgroups of plant 14-3-3s and focused atten tion on the influence of divergence in the

PAGE 130

116 extreme C-terminal regions as well as the subtle differences located in the mostly conserved binding groove. The answers to th ese questions transcend applications of 143-3s in plants. A seven member 14-3-3 gene family has been identified in humans, raising similar questions about the selectiv ity of isoform-specific binding and resultant specificity in function. Many of these isoforms have been impli cated with human disease, including certain cancers and neurodegenerative diseas es such as Cruezfeldt-Jakob disease, Alzheimers, and multiple sclerosis (Wilker et al. , 2004; van Everbroeck et al. , 2005; Teunissen et al. , 2005). For example, down regulation of 14-3-3 levels via either gene silencing or enhanced ubiquiti n-mediated degradation has been identified in cancers of the breast, lung, bladder, epithelial tissu e, and liver, among others (see Wilker et al. , 2004 for review). In normally functioning tissues , many studies have demonstrated this isoform’s upregulation and critic al role in maintaining the G2/M checkpoint in epithelial cells following DNA damage (Wilker et al. , 2004), explaining its si gnificance relative to cancer prevention. Furthermore, the14-3-3 isoforms and have been found to play important roles in cardiac function, while upregulation of 14-3-3 has been linked to the deactivation of the microtubule-associated protein, Tau, resulting in accumulation of neurofibrillary tangles that have been associated with Alzheimers (Wilker et al. , 2004). As the number of physiological and patholog ical human cell states that are found to involve specific 14-3-3 isoforms increases, questions concerning the use of these proteins as diagnostic and/or therap eutic agents are surfacing. Already, 14-3-3 isoforms , , , and are being used as CSF biomarkers for Creutzfeldt-Jakob disease (van Everbroek et al. , 2005). Along the same lines, it is easy to imagine the future use of assays which

PAGE 131

117 measure specific 14-3-3 isoform expression leve ls as a means for diagnosis of specific disease states, including thos e mentioned above. These assays may require a low degree of isoform selectivity, since specific tissues may be isolated a nd tested, excluding the influence of non-related 14-3-3 isoforms. Gi ven the wide range of cellular processes which elicit 14-3-3 regulation, however, the pr acticality of pharmaceutical regulation and control of specific 14-3-3 isof orm expression levels for the treatment of specific disease states would depend on a demonstration of hi ghly specific isoform function. As a result, the elucidation 14-3-3 structural features that lend to substrate selectivity and functional specificity remains a major focus of 14-3-3 re search in both animals and plants. The work presented in chapter 4 aimed to identi fy trends in 14-3-3 bi nding selectivity with respect to specific regions of structural variation among the isoforms themselves. This work represents a small contribution to the characterization of is oform-specific binding preferences that has yet to be accomplished, but offers a means through which further studies can be pursued.

PAGE 132

118 LIST OF REFERENCES Aitken, A.; Howell, S.; Jones, D.; Madrazo, J.; Patel, Y. -3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta: In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys motif.” Journal of Biological Chemistry 1995 , 270 , 5706-5709. Alpert, A.J. “Hydrophilic-int eraction chromatography for the separation of peptides, nucleic acids and other polar compounds.” Journal of Chromatography 1990 , 499 , 177-196. Amster, J. “Fourier tran sform mass spectrometry.” Journal of Mass Spectrometry 1996 , 31 , 1325-1337. Anderson, L.; Porath, J. “Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography.” Analytical Biochemistry 1986, 154 , 250-254. Annan, R.S.; Huddleston, M.J.; Verma, R. ; Deshaies, R.J.; Carr, S.A. “A multidimentional electrospray MS-based approach to phosphopeptie mapping.” Analytical Chemistry 2001 , 73, 393-404. Athwal, G.S.; Huber, J.L.; Huber, S.C. “B iological significance of divalent metal ion binding to 14-3-3 proteins in relationsh ip to nitrate reductase inactivation.” Plant Cell Physiology 1998 , 39 , 1065-1072. Athwal, G.S.; Huber, S.C. “Divalent cati ons and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction w ith phosphorylated nitrate reductase.” The Plant Journal 2002 , 29 , 119-129. Bachmann, M.; Huber, J.L.; Athwal, G.S.; W u, K.; Ferl, R.J.; Huber, S.C. -3-3 proteins associate with the regulatory phos phorylation site of sp inach leaf nitrate reductase in an isoform-specific manne r and reduce dephosphorylation of Ser-543 by endogenous protein phosphatases.” FEBS Letters 1996 , 398 , 26-30. Barlow Jr, R.B. “Circadian rhythms in the Limulus visual system.” Journal of Neuroscience 1983 , 3 , 856-870. Barlow Jr., R.B.; Chamberlain, S.C.; Levins on, J.Z. “Limulus brain modulates the structure and function of the lateral eyes.” Science 1980 , 210 , 1037-1039.

PAGE 133

119 Barnidge, D.R.; Dratz, E.A.; Martin, T.; Bonilla, L.E.; Moran, L.B.; Lindall, A. “Absolute quantification of the G-pr otein coupled receptor rhodopsin by LCMS/MS using proteolysis pr oduct peptides and syntheti c peptide standards.” Analytical Chemistry 2003 , 75 , 445-451. Battelle, B-A. “Circadian efferent input to Limulus eyes: anatomy, circuitry, and impact.” Microscopy Research and Technique 2002 , 58 , 345-355. Battelle, B-A.; Andrews, A.W.; Calman, B.G.; Sellers, J.R.; Greenburg, R.M.; Smith, W.C. “A Myosin III from Limulus eyes in a clock-regulated phosphoprotein.” The Journal of Neuroscience 1998 , 18 , 4548-4559. Battelle, B-A.; Evans, J.A.; Chamberlain, S.C. “Efferent fibers to Limulus eyes synthesize and release octopamine.” Science 1982 , 216 , 1250-1252. Battelle, B-A.; Robinson, N.A.; Kempler, K.E. “A circadian clock a nd light influence the distribution of myosin III in Limulus lateral eye photoreceptors.” Investigative Ophthalmology and Visual Science 2006 , 47 : ARVO eAbstract 5526 Battelle, B-A.; Sellers, J.R.; Kempler, K.E.; Mapel, G.M.; Fisler, H.; Stevens, S.; Yamashita, R.; Toth, J. “Limulus myosin III: Identification of a major PKA and autophosphorylation site and st udies of its actin binding and kinase activities.” Amer. Soc. For Cell Biol. 2004, Abstr. # 837. Beavis, R.C.; Chait, B.T. “Rapid, sensitive analysis of protein mixtures by mass spectrometry.” Proceedings of the National Academy for Sciences USA 1990 , 87 , 6873-6877. Becker, J.S.; Boulyga, S.F.; Pickhardt, C.; Becker, J.; Buddrus, S.; Przybylski, M. “Determination of phosphorus in small amounts of protein samples by ICP-MS.” Analytical and Bioanalytical Chemistry 2003 , 375 , 561-566. Bondarenko, P.V.; Chelius, D.; Shaler, T.A. “Identification and re lative quantitation of protein mixtures by enzymatic digesti on followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry.” Analytical Chemistry 2002 , 74 , 4741-4749. Boyle, W.J.; Van Der Geer, P.; Hunter, T. In Protein Phosphorylation ; Hunter, T.; Sefton, B.M., Eds.; Methods in Enzymol ogy Vol. 201; Academic Press: San Diego, New York, 1991 ; pp 110-149. Braselmann, S.; McCormick, F. “BCR and RA F form a complex in vivo via 14-3-3 proteins.” Journal of the European Mol ecular Biology Organization 1995 , 14 , 4839-4848. Burlingame, A.L. In Biological Mass Spectrometry , Burlingame, A.L., Ed.; Methods in Enzymology Vol. 402; Else vier Academic Press: Boston, 2005; Chapters 1 and 2.

PAGE 134

120 Carr, S.A.; Huddleston, M.J.; Bean, M.F. “S elective identification and differentiation of Nand O-linked oligosaccharides in gl ycoproteins by liquid chromatography-mass spectrometry.” Protein Science 1993 , 2 , 183-196. Cech, N.B.; Enke, C.G. “Practical implicati ons of some recent studies in electrospray ionization fundamentals.” Mass Spectrometry Reviews 2001 , 20 , 362 – 387. Chaga, G.S. “Twenty-five years of immobilized metal ion affinity chromatography: past, present, and future.” Journal of Biochemica l and Biophysical Methods 2001, 49 , 313-334. Chamberlain, S.C.; Barlow Jr, R.B. “Li ght and efferent activity control rhabdom turnover in Limulus photoreceptors.” Science 1979 , 206 , 361-363. Chamberlain, S.C.; Barlow Jr, R.B. “Transient membrane shedding in Limulus photoreceptors: control mechanis ms under natural lighting.” Journal of Neuroscience 1984 , 4 , 2729-2810. Chen, C.; Chen, Y. “Fe3O4/TiO2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using TiO2 surface-assisted laser desorption/ ionization mass spectrometry.” Analytical Chemistry 2005 , 77 , 5912-5919. Chernushevich, I.V.; Loboda, A.V.; Thomson, B.A. “An introduction to quadrupole-time of flight mass spectrometry.” Journal of Mass Spectrometry 2001 , 36 , 849-865. Dabdoub, A.; Payne, R. “Protein kinase C act ivators inhibit the visual cascade in Limulus ventral photoreceptors at an early stage.” The Journal of Neurosceince 1999 , 19 , 10262-10269. Dalal, S.N.; Schweitzer, C.M.; Gan, J.; DeCa prio, J.A. “Cytoplasmic localization of human cdc25C during interphase requir es intact 14-3-3 binding site.” Molecular and Cellular Biology 1999 , 19 , 4465-4479. Daugherty, C.J.; Rooney, M.F.; Miller, P.W.; Ferl, R.J. “Molecular organization and tissue-specific expression of an Arabidopsis 14-3-3 gene.” Plant Cell 1996 , 8 , 1239-1248. Dawson, R.M.C. In Data for Biomedical Research. Clarendon Press: Oxford, 1959. Dos , A.; Burnside, B. “Cloning and chromo somal localization of a human class III myosin.” Genomics 2000 , 67 , 333-342. Dos , A.; Burnside, B. “A class III myosin expressed in the retina is a potential candidate for Bardet-Biedl syndrome.” Genomics 2002 , 79 , 621-624. Edwards, S.C.; Battelle, B-A. “Octopamine and cAMP-stimulated phosphorylation of a protein in Limulus ventral and lateral eyes.” Journal of Neuroscience 1987 , 7 , 2811-2820.

PAGE 135

121 Enke, C.G. “A predictive m odel for the matrix and analyt e effects in electrospray ionization of singly-char ged ionic analytes.” Analytical Chemistry 1997 , 69 , 4885– 4893. van Everbroeck, B.; Boons, J.; Cras, P. “Cer ebrospinal fluid biomarkers in CreutzfeldtJakob disease.” Clinical Neurology and Neurosurgery 2005 , 107 , 355-360. Fenn, J.B.; Mann, M.; Meng, C.K.; Wong, S.F. ; Whitehouse, C.M. “Electrospray ionization for mass spectrometr y of large biomolecules.” Science 1989 , 246 , 64-71. Ferl, R.J. -3-3 proteins: regula tion of signal-induced events.” Physiologia Plantarum 2004 , 120 , 173-178. Ficarro, S.B.; McCleland, M.L.; Stukenberg, P.T.; Burke, D.J.; Ross, M.M.; Shabanowitz, J.; Hunt, D.F.; White, F. M. “Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cervisiae .” Nature Biotechnology 2002 , 20 , 301-305. Flora, J.W.; Muddiman, D.C. “Selectiv e, sensitive, and rapid phosphopeptide identification in enzymatic digests using ESI-FTICR-MS with infrared multiphoton dissociation.” Analytical Chemistry 2001 , 73 , 3305-3311. Flora, J.W.; Muddiman, D.C. “Gas-phase ion unimolecular dissociation for rapid phosphopeptide mapping by IRMPD in a penning ion trap: an energentically favored process.” Journal of the American Chemical Society 2002 , 124 , 65466547. Furch, M.; Geeves, M.; Manstein, D.J. “M odulation of actin affinity and actomyosin adenosine triphosphatase by charge cha nges in the myosin motor domain.” Biochemistry 1998 , 37 , 6317-6326. Gaberc-Porekar, V.; Menart, V. “Perspectives of immobilized-metal affinity chromatography.” Journal of Biochemical and Biophysical Methods , 2001, 49 , 335-360. Gerber, S.A.; Rush, J.; Stemman, O.; Kirs chner, M.W.; Gygi, S.P. “Absoltue quantification of proteins and phosphoprot eins from cell lysates by tandem MS.” Proceedings of the National Academy for Sciences 2003 , 100 , 6940-6945. Gomez, A.; Tang, K. “Charge and fission of droplets in electrostatic sprays.” Physics of Fluids 1994 , 6 , 404-414. Goodlett, D.R.; Keller, A.; Watts, J.D.; New itt, R.; Yi, E.C.; Purvine, S.; Eng, J.K.; von Haller, P.; Aebersold, R.; Kolker, E. “D ifferential stable isotope labeling of peptides for quantitation a nd de novo sequence derivation.” Rapid Communications in Mass Spectrometry 2001 , 15 , 1214-1221.

PAGE 136

122 Grayson, M.A., ed. Measuring Mass: From Po sitive Rays to Proteins . Chemical Heritage Press: Philadelphia, 2002 . Gygi, S.P.; Rochon, Y.; Franza, B.R.; Aebersol d, R. “Correlation between protein and mRNA abundance in yeast.” Molecular & Cellular Biology 1999 , 19 , 1720-1730. Gygi, S.P.; Rist, B.; Gerber, S.A.; Turecek, F. ; Gelb, M.H.; Aebersold, R. “Quantitatve analysis of complex protein mixtures using isotope-coded affinity tags.” Nature Biotechnology 1999, 17 , 994-999. Hakansson, K.; Chalmers, M.J.; Quinn, J.P.; McFarland, M.A.; Hendrickson, C.L.; Marshall, A.G. “Combined electron capture and infrared multiphoton dissociation for multistage MS/MS in a Fourier transform ion cyclotron resonance mass spectrometer.” Analytical Chemistry 2003 , 75 , 3256-3262. van Heusden, G. Paul H. -3-3 proteins: regu lators of numerous eukaryotic proteins.” International Union of Biochemi stry and Molecular Biology Life 2005 , 57 , 623629. van Heusden, G.P.; van der Zanden, A.L.; Ferl, R.J.; Steensma, H.Y. “Four Arabidopsis thaliana 14-3-3 protein isoforms can complement the lethal yeast bmh1 bmh2 double disruption.” Federation of European Biochemical Societies Letters 1996 , 391 , 252-256. Huber, S.C.; Bachmann, M.; Huber, J.L. “Post-translational re gulation of nitrate reductase activity: a role for Ca2+ and 14-3-3 proteins.” Trends in Plant Science 1996 , 1 , 432-438. Ikeguchi, Y.; Nakamura, H. “Determinati on of organic phosphates by column-switching high performance anion-exchange chroma tography using on-line preconcentration on titania.” Analytical Sciences 1997 , 13, 479-483. Ikeguchi, Y.; Nakamura, H. “Selective enrichment of phospholipids by titania.” Analytical Sciences 2000 , 16 , 541-543. Jinks, R.N.; White, R.H.; Chamberlain, S.C. “Dawn, diacylglycerol, calcium, and protein kinase C – The retinal wrecking crew. A signal transduction cascade for rhabdom shedding in Limulus eye.” Journal of Photochemistry and Photobiology B 1996 , 35 , 45-52. Kaplan, E.; Barlow Jr, R.B. “Circadian clock in Limulus brain increases responses and decreases noise of retinal photoreceptors.” Nature 1980 , 286 , 393-395. Karas, M.; Hillenkamp, F. “Laser desorpti on ionization of proteins with molecular masses exceeding 10,000 Da.” Analytical Chemistry 1988 , 60 , 2299-2301. Kaupp, U.B.; Malbon, C.C.; Battelle, B-A.; Brown, J.E. “Octopamine stimulated rise in cAMP in Limulus ventral photoreceptors.” Vision Research 1982 , 22 , 1503-1506.

PAGE 137

123 Kebarle, P.; Ho, Y.; In On the Mechanism of Electrospray Mass Spectrometry ; Cole, R.B., ed.; Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, & Applications; Jo hn Wiley & Sons, Inc.: New York, 1997 ; 3-64. Keihl, D.E.; Julian, R.K.; Kennington, A.S. “Electrospray ionization mass spectrometry with in-source collision-i nduced dissociation or mone nsin factors and related metabolites.” Rapid Communications in Mass Spectrometry 1998 , 12 , 903-910. Kempler, K.E.; Toth, J.; Yamashita, R.; Mape l, G.; Robinson, K.; Cardasis, H.; Stevens Jr., S.; Sellers, J.R.; Battelle, B-A. “ Limulus myosin III: identification of protein kinase A and autophosphorylation sites a nd characterization of its kinase, actin binding, and motor properties.” Submitted for publication in Journal of Biological Chemistry 2006 . Kier, C.K.; Chamberlain, S.C. “Dual cont rols of screening pigment movement in photoreceptors of the Limulus lateral eye: circadian e fferent input and light.” Visual Neuroscience 1989 , 4 , 237-255. Knox, J.H.; Kaur, B.; Millward, G.R. “Struc ture and performance of porous graphitic carbon in liquid chromatography.” Journal of Chromatography 1986 , 352 , 3-25. Komaba, S.; Inoue, A.; Maruta, S.; Hosoya, H.; Ikebe, M. “Determination of human myosin III as a motor protein having protein kinase activity.” The Journal of Biological Chemistry 2003 , 278 , 21352-21360. Krysan, P.J.; Young, J.C.; Tax, F.; Sussman, M. R. “Identification of transferred DNA insertions within Arabidopsis genes involved in signa l transduction and ion transport.” Proc. Natl. Acad. Sci. USA 1996 , 93 , 8145-8150. Kumler, W.D.; Eiler, J.J. “The acid streng th of mono and di-esters of phosphoric acid. The n-alkyl esters from methyl to butyl, th e esters of biological importance, and the natural guanidine phosphoric acid.” Journal of the American Chemical Society 1943 , 65 , 2355-2361. Kuromori, T.; Yamamoto, M. “Members of the Arabidopsis 14-3-3 gene family transcomplement two types of defects in fission yeast.” Plant Science 2000 , 158 , 155161. Larsen, M.R.; Thingholm, T.E.; Jensen, O.N.; Roepstorff, P.; Jorgensen, T.J.D. “Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.” Molecular & Cellular Proteomics 2005 , 4 (7), 873-886. Laskin, J.; Futrell, J.H. “Collisional ac tivation of peptide ions in FT-ICR mass spectrometry.” Mass Spectrometry Reviews 2003 , 22 , 158-181. Lill, J. “Proteomic tools for quantitation by mass spectrometry.” Mass Spectrometry Reviews 2003 , 22 , 182-194.

PAGE 138

124 Little, D.P.; Speir, J.P.; Senko, M.W.; O’ Connor, P.B.; McLafferty, F.W. “Infrared multiphoton dissociation of large multip ly charged ions for biomolecular sequencing.” Analytical Chemsitry 1994 , 66 , 2809-2815. Liu, D.; Bienkowska, J.; Petosa, C.; Collier, R.J.; Fu, H.; Liddington, R. “Crystal structure of the zeta isofor m of the 14-3-3 protein.” Nature 1995 , 376 , 191-194. Lu, G.; Sehnke, P.C.; Ferl, R.J. “Phosphoryla tion and calcium binding properties of an Arabidopsis GF14 brai n protein homolog.” Plant Cell 1994 , 6 , 501-510. March, R.E.; Todd, J.F.J. Quadrupole Ion Trap Mass Spectromety, 2nd ed. ; John Wiley & Sons, Inc.: Hoboken, N.J., 2005 . Marshall, A.G.; Hendrickson, C.L.; Jackson, G.S. “Fourier transform ion cyclotron resonance mass spectrometry: a primer.” Mass Spectrometry Reviews 1998 , 17 , 135. Martin, H.; Patel, Y.; Jones, D.; Howell, S.; Robinson, K.; Aitken, A. “Antibodies against the major isoforms of 14-3-3 protein. An antibody specific for the Nacetylated amino terminus of a protein.” FEBS Letters 1993 , 331 , 296-303. McLachlin, D.T.; Chait, B.T. “Analysis of phosphorylated proteins and peptides by mass spectrometry.” Current Opinion in Chemical Biology 2001 , 5 , 591-602. Minke, B.; Rubinstein, C.T.; Sahly, I.; Bar-N achum, S.; Timberg, R.; Selinger, Z. “Phorbol ester induces photorecep tor-specific degeneration in a Drosophila mutant.” Proceeding of the National Academy of Science USA 1990 , 87 , 113-117. Moore, B.W.; Perez, V.J. “Specific acidic pr oteins of the nervous system.” In: Carlson, F. (ed) Physiological and Biochemical Aspects of Nervous Integration 1967 , Prentice Hall, Woods Hole, MA, 343-359. Moore, D.D.; Sefton, B.M. In Current Protocols in Molecular Biology ; John Wiley & Sons, Inc.; 1996 , Supplement 33; 18.5.1-18.5.9. Moore, D.D.; Sefton, B.M. In Current Protocols in Molecular Biology ; ; John Wiley & Sons, Inc.; 2000 , Supplement 50.; 18.6.1-18.6.18. Moore, D.D; Sefton, B.M. In Current Protocols in Molecular Biology ; John Wiley & Sons, Inc., 2004 , Supplement 68; 18.1.1-18.1.5. Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. “Quantitation and facilitated de novo sequencing of proteins by isotopic N-te rminal labeling of peptides with a fragmentation-directing moiety.” Analytical Chemistry 2002 , 72 , 4047-4057. Muszynska, G.; Anderson, L.; Porath, J. “S elective adsorption of phosphoproteins on gel-immobilized ferric chelate.” Biochemistry 1986, 25 , 6850-6853.

PAGE 139

125 Obsil, T.; Ghirlando, R.; Klein, D.C.; Ganguly, S. ; Dyda, F. “Crystal structure of the 143-3 :serotonin N-acetyltransferase complex: A role for scaffolding in enzyme regulation.” Cell 2001 , 105 , 257-267. Oda, Y.; Huang, K.; Cross, F.R.; Cowburn, D.; Chait, B.T. “Accurate quantitation of protein expression and site-s pecific phosphorylation.” Proceedings of the National Academy for Sciences USA 1999 , 96 , 6591-6596. Ong, S.E.; Blagoev, B.; Kratchmarova, I.; Kr istensen, D.B.; Steen, H.; Pandey, A.; Mann, M. “Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.” Molecular and Cellular Proteomics 2002 , 1 , 376-386. Paul, A-L.; Sehnke, P.C.; Ferl, R.J. “Isofo rm-specific subcellula r localization among 143-3 proteins in Arabidopsis seems to be driven by client interactions.” Molecular Biology of the Cell 2005 , 16 , 1735-1743. Pearson, R.G. In Hard and soft acids and bases. Rearson, R.G., Ed.; Hutchington & Ross: Stroudsburg, PA, 1973, p. 53-59, 67-85. Peng, J.; Schwartz, D.; Elias, J.E.; Thoreen, C.C.; Cheng, D.; Marsischky, G.; Roelofs, J.; Finley, D.; Gygi, S.P. “A proteomi cs approach to understanding protein ubiquitination.” Nature Biotechnology 2003 , 21 , 921-926. Peters, E.C.; Brock, A.; Ficarro, S.B. “Exploring the phosphoproteome with mass spectrometry.” Mini-Reviews in Medicinal Chemistry 2004 , 4 , 313-324. Peters, E.C.; Horn, D.M.; Tully, D.C.; Brock, A. “A novel multifunctional labeling reagent for enhancing protein charac terization with mass spectrometry.” Rapid Communications in Mass Spectrometry 2001 , 15 , 2387-2392. Pinkse, M.W.H.; Uitto, P.M.; Hilhorst, M.J.; Oo ms, B.; Heck, A.J.R. “Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2DnanoLC-ESI-MS/MS and titanium oxide precolumns.” Analytical Chemistry 2004 , 76 , 3935-3943. Porath, J.; Carlsson, J.; Olsson, I.; Belfrage G. “Metal chelate affinity chromatography, a new approach to protein fractionation.” Nature 1975 , 258 , 598-599. Posewitz, M.C.; Tempst, P. “Immobilized gallium(III) affinity chromatography of phosphopeptides.” Analytical Chemistry 1999 , 71 , 2883-2892. Qui, Y.; Sousa, E.A.; Hewick, R.M.; Wang, J.H. “Acid-labile isot ope-coded extractants: A class of reagents for quantitative mass spectrometric analysis of complex protein mixtures.” Analytical Chemistry 2002 , 74 , 4969-4979.

PAGE 140

126 Raska, C.S.; Parker, C.E.; Dominski, Z.; Marzluff, W.F.; Glish, G.L.; Pope, R.M.; Borchers, C.H. “Direct MALDI-MS/MS of phosphopeptides affinity bound to immobilized metal ion affin ity chromatography beads.” Analytical Chemistry 2002 , 74 , 3429-3433. Roach, P.J.; Wang, Y. In Protein Phosphorylation ; Hunter, T., Sefton, B.M., Eds.; Methods in Enzymology 201; Academic Press: San Diego, New York, 1991; pp 169-185. Rosenquist, M.; Sehnke, P.; Ferl, R.J.; Sommarin, M.; Larsson, C. “Evolution of the 143-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?” Journal of Molecular Evolution 2000 , 51 , 446-458. Ross, P.L.; Huang, Y.N.; Marchese, J.N. ; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D.J. “Multiplexed protein quantitation in Saccharomyces cervisiae using amine-reactive isobaric tagging reagents.” Mollecular and Cellular Proteomics 2004 , 3 , 1154-1169. Ruse, C.I.; Willard, B.; Jin, J.P.; Hass, T.; Kinter, M.; Bond, M. “Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry.” Analytical Chemistry 2002 , 74 , 16581664. Sacunas, R.B.; Papuga, M.O.; Malone, M.A.; Pearson, Jr., A.C.; Marjanovic, M.; Stroope, D.G.; Weiner, W.W.; Chamberlai n, S.C.; Battelle, B-A. “Multiple mechanims of rhabdom sheddi ng in the lateral eye of Limulus polyphemus .” The Journal of Comparative Neurology 2002 , 449 , 26-42. Schultz, T.F.; Medina, J.; Hill, A.; Quatrano, R.S. -3-3 proteins are part of an abscisic acid-VIVIPAROUS1 (VP1) response complex in the Em promoter and interact with VP1 and EmBP1.” Plant Cell 1998 , 10 , 837-847. Sefton, B.M. In Current Protocols in Molecular Biology ; John Wiley & Sons, Inc.; 1997 ; Supplement 40; 18.3.1-18.3.8. Sehnke, P.C.; Chung, H.J.; Wu, K.; Ferl, R.J. “Regulation of star ch accumulation by granule-associated plan t 14-3-3 proteins.” Proceedings of the National Acadamy for Sciences USA 2001 , 98 , 765-770. Sehnke, P.C.; Henry, R.; Cline, K.; Ferl, R.J. “Interaction of a plant 14-3-3 protein with the signal peptide of a th ylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma.” Plant Physiology 2000 , 122 , 235-242. Sehnke, P.C.; Laughner, B.; Cardasis, H.; Powell, D.; Ferl, R.J. “Exposed loop domains of complexed 14-3-3 proteins contribute to structural diversity and functional specificity.” Plant Physiology 2006 , 140 , 647-660.

PAGE 141

127 Sehnke, P.C.; Rosenquist, M.; Alsterfjord, M. ; DeLille, J.; Sommarin, M.; Larsson, C.; Ferl, R.J. “Evolution and isoform speci ficity of plant 14-3-3 proteins.” Plant Molecular Biology 2002 , 50 , 1011-1018. Sekimoto, T.; Fukumoto, M.; Yoneda, Y. “ 14-3-3 suppresses the nuc lear localization of threonine 157-phosphorylated p27Kipl.” Journal of the European Molecular Biology Organization 2004 , 23 , 1934-1942. Sineshchekova, O.O.; Cardasis, H.; Severace, E.G.; Smith, W.C.; Battelle, B.A. “Sequential phosphorylation of vi sual arrestin in intact Limulus photoreceptors: Identification of a highl y light-regulated site.” Visual Neuroscience 2004 , 21 , 715724. Sharma, S.; Argarwal, G.P. “Interactions of proteins with immobilized metal ions: role of ionic strength and pH.” Journal of Colloid and Interface Science 2001 , 243 , 6172. Steinberg, T.H.; Agnew, B.J.; Gee, K.R. ; Leung, W.L.; Goodman, T.; Schulenberg, B.; Hendrickson, J.; Beechem, J.M.; Haugland, R.P.; Patton, W.F. “Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology.” Proteomics 2003 , 3 , 1128-1144. Subramanian, R.R.; Zhang, H.; Wang, H.; Ichijo , H.; Miyashita, T.; Fu, H. “Interaction of apoptosis signal-regulati ng kinase 1 with isoforms of 14-3-3 proteins.” Experimental Cell Research 2004 , 294 , 581-591. Testerink, C.; van der Meulen, R.M.; Oppe dijk, B.J.; de Boer, A.H.; HeimovaaraDijkstra, S.; Kijne, J.W.; Wang, M. “Di fferences in spatial expression between 143-3 isoforms in germinating barley embryos.” Plant Physiology 1999 , 121 , 81-88. Teunissen, C.E.; Dijkstra, C.; Polman, C. “Biological markers in CSF and blood for axonal degeneration in multiple sclerosis.” Lancet Neurology 2005 , 4 , 32-41. Tonner, D.S.; McMahon, T.B. “Consecutive infrared multiphoton dissociations in a Fourier transform ion cyclotron resonance mass spectrometer.” Analytical Chemistry 1997 , 69 , 4735-4740. Trinidad, J.C.; Specht, C.G.; Thalhammer, A.; Schoepfer, R.; Burlingame, A.L. “Comprehensive identificati on of phosphorylatio n sites in post synaptic density preparations.” Molecular and Cellular Proteomics 2006 , 5 , 914-922. Truong, A.B.; Masters, S.C.; Yang, H.; Fu, H. “Role of the 14-3-3 C-terminal loop in ligand interaction.” Protein 2002 , 49 , 321-325. Ueda, E.K.M.; Gout P.W.; Morganti L. “Curre nt and prospective applications of metal ion-protein binding.” Journal of Chromatography A 2003 , 98 , 1-23.

PAGE 142

128 Vacratsis, P.O.; Phinney, B.S.; Gage, D.A.; Gallo, K.A. “Identification of in vivo phosphorylation sites of MLK3 by ma ss spectrometry and phosphopeptide mapping.” Biochemistry 2002 , 41 , 5613-5624. Visconti, S.; Camoni, L.; Fullone, M.R.; Lalle , M.; Marra, M.; Aducci, P. “Mutational analysis of the interaction between 14-3 -3 proteins and plant plasma membrane H+-ATPase.” The Journal of Biological Chemistry 2003 , 278 , 8172-8178. Walsh, T.; Walsh, V.; Vreugde, S.; Hertzano, R.; Shahin, H.; Haika, S.; Lee, M.K.; Kanaan, M.; King, M-C.; Avraham, K.B. “F rom flies eyes to our ears: mutations in a human class III myosin cause progressive nonsyndromic hearing loss DFNB30.” Proceedings of the National Acadamy for Sciences USA 2002 , 99 , 7518-7523. Wang, H.; Zhang, H.; Liddington, R.; Fu, H. “Mutations in the hydrophobic surface of an amphipathic groove of 14-3-3zeta disrupt its interaction with Raf-1 kinase.” The Journal of Biological Chemistry 1998 , 273 , 16297-16304. Watanabe, M.; Isobe, T.; Ichimura, T.; Kuwa no, R.; Takahashi, Y.; Kondo, H.; Inoue, Y. “Molecular cloning of rat cDNAs for the zeta and theta s ubtypes of 14-3-3 protein and differential distributions of their mRNAs in the brain.” Brain Research. Molecular Brain Research. 1994 , 25 , 113-121. Wilker, E.; Yaffe, M.B. -3-3 Proteins -a focus on cancer and human disease.” Journal of Molecular and Cellular Cardiology 2004 , 37 , 633-642. Wilm, M.; Mann, M. “Analytical propertie s of the nanoelectrospray ion source.” Analytical Chemistry 1996, 68 , 1-8. Yan, J.X.; Packer, N.H.; Gooley, A.A.; Williams, K.L. “Protein phosphorylation: technologies for the identific ation of phosphoamino acids.” Journal of Chromatography A 1998 , 808 , 23-41. Yao, X.; Freas, A.; Ramirez, J.; Demirev, P.; Fenselau, C. “Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus.” Analytical Chemistry, 73 , 2836-2842. Zhang, L.; Wang, H.; Liu, D.; Liddington, R.; F u, H. “Raf-1 kinase and exoenzyme S interact with 14-3-3 ze ta through a common site involving lysine 49.” Journal of Biological Chemistry 1997 , 272 , 13717-13724. Zhang, L.; Wang, H.; Masters, S.C.; Wang, B.; Ba rbieri, J.T.; Fu, H. “Residues of 14-33 required for activatio n of exoenzyme S of Pseudomonas aeruginosa. ” Biochemistry 1999 , 38 , 12159-12164. Zook, D.R.; Bruins, A.P. “On cluster ions , ion transmission, and linear dynamic range limitations in electrospray (ionspray) mass spectrometry.” International Journal of Mass Spectrometry and Ion Processes 1997 , 162 , 129-147.

PAGE 143

129 Zubarev, R.A. “Reactions of polypeptide i ons with electrons in the gas phase.” Mass Spectrometry Reviews 2003 , 22 , 57-77.

PAGE 144

130 BIOGRAPHICAL SKETCH Helene L. Cardasis attended Fairleigh Di ckinson University in Madison, NJ, from August of 1997 to May of 2001. During the summ er of 2000, she took part in a National Science Foundation – Research Experience fo r Undergraduates (NSF-REU) internship at the University of Florida’s Whitney Laborator y in St. Augustine, FL. There she studied the dynamics of arrestin phosphorylation in th e photoreceptors of horseshoe crabs. Helene earned a Bachelor of Science degree with a major in chemistry and minors in math and biology in 2001, and went on to pur sue graduate work that summer at the University of Florida’s Department of Chemis try in Gainesville, FL. During her graduate school career, she worked on mass spectrome tric analysis of phosphorylation-based signal transduction in two unique biological systems, that of the Myosin III from the photoreceptors of horseshoe crab s, and 14-3-3 proteins from Arabidopsis thaliana . Following the successful defense of her diss ertation in the June of 2006, Helene will pursue a post doctoral appointment at the Ne w York University School of Medicine, where she will continue to utilize mass spectro metry to tackle biomedical questions.