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Maldi Imaging of Myelin Basic Protein in Traumatic Brain Injury

Permanent Link: http://ufdc.ufl.edu/UFE0044765/00001

Material Information

Title: Maldi Imaging of Myelin Basic Protein in Traumatic Brain Injury
Physical Description: 1 online resource (48 p.)
Language: english
Creator: Mangaonkar, Manasi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: mbp -- msi -- tbi
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Traumatic braininjury (TBI) is damage to the brain caused by a jolt or penetration of anobject into the head. The symptoms of TBI (e.g., headache, unconsciousness,internal bleeding, loss of memory, or concussions) depend on the severity ofthe injury. Severe cases of TBI can also result in the death of an individual. TBIcauses a substantial number of deaths and permanent disabilities in the UnitedStates. Recent data has shown that approximately 1.7 million people suffer fromTBI annually. Neurotrauma, whichoccurs after TBI, promotes degradation of proteins in the brain. Degradationproducts of proteins following TBI have been used to study the post injurymechanism; thus, degradation products may also be used as injury biomarkers. Aspart of the nervous system, myelin membranes protect and insulate the neuronsand aid in transmission of signals between neurons. These myelin membranes arecomposed of lipid and protein layers. Amongst all the myelin membrane proteins,myelin basic protein (MBP), which is present in various isoforms, is the mostabundant. Following TBI, MBP is subjected to degradation. Mass spectrometric imaging (MSI) is a powerfulanalytical tool used to study the localization of an analyte in tissue. Thiswork utilizes MSI to localize MBP in both the control and TBI brain. Two isoformsof MBP (14 kDa and 18 kDa) are observed in the white matter, especially in thecorpus callosum and hippocampus region of the rat brain. The intensities ofthese two isoforms of MBP decrease in the injured brain section, indicating thedegradation or breakdown of MBP following TBI.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Manasi Mangaonkar.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Yost, Richard A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044765:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044765/00001

Material Information

Title: Maldi Imaging of Myelin Basic Protein in Traumatic Brain Injury
Physical Description: 1 online resource (48 p.)
Language: english
Creator: Mangaonkar, Manasi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: mbp -- msi -- tbi
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Traumatic braininjury (TBI) is damage to the brain caused by a jolt or penetration of anobject into the head. The symptoms of TBI (e.g., headache, unconsciousness,internal bleeding, loss of memory, or concussions) depend on the severity ofthe injury. Severe cases of TBI can also result in the death of an individual. TBIcauses a substantial number of deaths and permanent disabilities in the UnitedStates. Recent data has shown that approximately 1.7 million people suffer fromTBI annually. Neurotrauma, whichoccurs after TBI, promotes degradation of proteins in the brain. Degradationproducts of proteins following TBI have been used to study the post injurymechanism; thus, degradation products may also be used as injury biomarkers. Aspart of the nervous system, myelin membranes protect and insulate the neuronsand aid in transmission of signals between neurons. These myelin membranes arecomposed of lipid and protein layers. Amongst all the myelin membrane proteins,myelin basic protein (MBP), which is present in various isoforms, is the mostabundant. Following TBI, MBP is subjected to degradation. Mass spectrometric imaging (MSI) is a powerfulanalytical tool used to study the localization of an analyte in tissue. Thiswork utilizes MSI to localize MBP in both the control and TBI brain. Two isoformsof MBP (14 kDa and 18 kDa) are observed in the white matter, especially in thecorpus callosum and hippocampus region of the rat brain. The intensities ofthese two isoforms of MBP decrease in the injured brain section, indicating thedegradation or breakdown of MBP following TBI.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Manasi Mangaonkar.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Yost, Richard A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044765:00001


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1 MALDI IMAGING OF MYELIN BASIC PROTEIN IN TRAUMATIC BRAIN INJURY By MANASI DILIP MANGAONKAR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Manasi D. Mangaonkar

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3 To my parents and Naren

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4 ACKNOWLEDGMENTS There are several people I would like to thank for their support in pursuing my thank Dr. Powell for accepting me as his student into the group. His guidance created an environment where I co uld put together ideas to work. I would also like to thank Dr. Yost for his guidance. His sub g roup and group meetings have provided me with more knowledge in the fiel d of mass spectrometric research group for their support. Special thanks go out to Rob and Noelle for their advice and help I want to thank my collabor ator, Dr. Kevin Wang from the Mc Knight Brain Institute at the University of Florida for his guidance and allowing me to perform preliminary studies in his lab. I also want to acknowledge Dr. Zhiqun Zhang and Dr. Firas Koibeissy for their efforts in arrangi ng for the brain samples. Thank you to my parents for encouraging and supporting me and giving me the opportunity to pursue graduate studies I would also like to thank my husband, Naren, for helping me through some difficult times. Without their love and support t his would not have been possible. Finally, I thank my frien ds and family for their support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGUR ES ................................ ................................ ................................ .......... 6 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 10 Background ................................ ................................ ................................ ............. 10 Sample Preparation ................................ ................................ ................................ 11 Tissue Handling and Storage ................................ ................................ ........... 11 Tissue Sectioning a nd Mounting ................................ ................................ ...... 11 Tissue Washing and Application of Matrix ................................ ........................ 12 Instrumentation ................................ ................................ ................................ ....... 13 Matrix Assisted Laser Desorption Ionization (MALDI) ................................ ...... 13 Time of Flight (TOF) Mass Analyzer ................................ ................................ 14 TOF TOF Mass Spectrom eter ................................ ................................ .......... 16 Peptide Fragmentation and Nomenclature ................................ ....................... 17 2 MALDI IMAGING OF MYELIN BASIC PROTEIN IN TRAUMATIC BRAIN INJURY ................................ ................................ ................................ ................... 21 Background ................................ ................................ ................................ ............. 21 Experimental ................................ ................................ ................................ ........... 23 Materials and Chemicals ................................ ................................ .................. 23 MBP Standard ................................ ................................ ................................ .. 23 Samples ................................ ................................ ................................ ........... 23 Gel Electrophoresis ................................ ................................ .......................... 24 Tissue Preparation ................................ ................................ ........................... 24 Instrumentation ................................ ................................ ................................ 25 Data Processing ................................ ................................ ............................... 26 Results and Discussion ................................ ................................ ........................... 26 Brain Lysate Analysis ................................ ................................ ....................... 26 Direct Brain Analysis ................................ ................................ ........................ 27 3 CONCLUSIONS AND FUTURE WORK ................................ ................................ 44 LIST OF REFERENCES ................................ ................................ ............................... 46 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 48

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6 LIST OF FIGURES Figure page 1 1 Schmetic diagram of linear time of flight (TOF) mass spectrometer. Adapted from Watson, J.; Sparkman, D., Introduction to M ass Spectrometry Instrumentation, Applications and Strategies for data Interpretation John Wiley and sons Ltd, 4 th Edition, 2008. [14] ................................ ............................ 18 1 2 Schmetic diagram of reflectron time of flight (T OF) mass spectrometer. Adapted from Watson, J.; Sparkman, D., Introduction to Mass Spectrometry Instrumentation, Applications and Strategies for data Interpretation John Wiley and sons Ltd, 4 th Edition, 2008. [14] ................................ ............................ 19 1 3 Nomenclature for peptide fragments generated by tandem mass spectrometry. ................................ ................................ ................................ ...... 20 2 1 Schmatic representation for linear mode operation on the AB SCIEX 5800 MALDI TOF TOF instrument. ................................ ................................ ............. 29 2 2 Schmatic representation for reflector mode operation on the AB SCIEX 5800 MALDI TOF TOF instrument. The ion path for the product ions from MS 2 is shown in orange. ................................ ................................ ................................ 30 2 3 MS spectrum of MBP standard. In this spectrum, the 18 kDa isoform of MBP forms dominant peak. ................................ ................................ ......................... 31 2 4 Image of the 1 dimensiona l gel electrophoresis for the brain lysates from different regions and models; human brain, ipsilateral cortex nave (IC N), ipsilateral hippocampus nave (IH N), ipsilateral cortex injured (IC I), ipsilateral hippocampus injured (IH I). A molecular weight marker (MWM) was used as a reference. ................................ ................................ ................... 32 2 5 In silico digestion of MBP using Protein Prospector. ................................ .......... 33 2 6 MS spectrum of trypsin digestion of MBP band from control human brain lysate. ................................ ................................ ................................ ................. 34 2 7 A) MS spectrum of trypsin digestion of MBP band from injured ipsilateral hippocampus (IH I) rat brain lysate. B) The zoom in spec trum displays the mass range m/z 700 750, to show m/z 726 and m/z 728 distinctly. ................... 34 2 8 MS 2 spectrum of m/z 698 from control human brain lysate. ............................... 35 2 9 MS 2 spectrum of m/z 728 from IH I rat brain lysate. ................................ ........... 35 2 10 MS 2 spectrum of m/z 726 from control human brain lysate. ............................... 36

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7 2 11 MS 2 spectrum of m/z 726 from IH I rat brain lysate. ................................ ........... 36 2 12 MS 2 spectrum of m/z 1046 from control human brain lysate. ............................. 37 2 13 MS 2 spectrum of m/z 1046 from IH I rat brain lysate. ................................ ......... 37 2 14 MS 2 spectrum of m/z 1460 from control human brain lysate. ............................. 38 2 15 Average mass spectrum from the coronal section of the control rat brain. ......... 39 2 16 MSI images of coronal section of the control rat brain. The two images illustrate the locali zation of different isoforms of MBP. Image A shows the distribution of 14 kDa isoform of MBP and image B shows the distribution of 18 kDa isoform of MBP. All intensities are normalized to the mean intensity of each pixel with baseline correction. ................................ ................................ .... 40 2 17 Rat MBP sequence for 14 kDa isoform. ................................ ............................. 41 2 18 Rat MBP sequence for 18 kDa isoform. ................................ ............................. 41 2 19 MSI images of coronal section of the TBI rat brain. Image A shows the distribution of 14 kDa isoform of MBP and image B shows the distribution of 18 kDa isoform of MBP. All intensities are normalized to the mean intensity of each p ixel with baseline correction. ................................ ................................ .... 42 2 20 MSI images of coronal section of the TBI rat brain showing the suspected breakdown products of MBP. All intensities are normalized to the mean intensity of each pixel with baseline correction. ................................ .................. 43

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8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MALDI IMAGING OF MYELIN BASIC PROTEIN IN TRAUMATIC BRAIN INJURY By Manasi D. Mangaonkar August 2012 Chair: Richard A. Yost Major: Chemistry Traumatic brain injury (TBI) is damage to the brain caused by a jolt or penetration of an object into the head. T he symptoms of TBI (e.g., headache, unconsciousness, internal bleeding, loss of memory, or concussions) depend on the severity of the injur y. Severe cases of TBI can also result in the death of an individual. TBI causes a substantial number of deaths and p ermanent disabilities in the United States. Recent data has shown that approximately 1.7 million people suffer from TBI annually. Neurotrauma which occurs after TBI promotes degradation of proteins in the brain. Degradation products of proteins following TBI have been used to study the post in j ury mechanism ; thus degradation products may also be used as injury biomarkers. As part of the nervous system, m yelin membranes protect and insulate the neurons and aid in transmission of signals between neurons. T he se myelin membranes are composed of lipid and protein layers. Among st all the myelin membrane proteins, myelin basic protein (MBP) which is present in various isoforms, is the most abundant. Following TBI, MBP is subjected to degradation

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9 Mass spectrom etric imaging ( MSI ) is a powerful analytical tool used to study the localization of an analyte in tissue. This work utilizes MSI to localiz e MBP in both the control and TBI brain. Two isoforms of MBP (14 kDa and 18 kDa) are observed in the white matter, es pecially in the corpus callosum and hippocampus region of the rat brain. The intensities of these two isoforms of MBP dec r ease in the injured brain section indicat ing the degradation or breakdown of MBP following TBI.

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10 CHAPTER 1 INTRODUCTION Background P roteomics studies are rapidly evolv ing to provide the opportunity to identify and characterize protein profiles of highly complex samples. Proteins are the most versatile macromolecules act ing as catalysts, messengers, transporters or building blocks in the living system [1] The degradation products of proteins, result ing from damage to the intact protein can be studied as biomarkers in a particular disease or disorder. For example, the increase in the breakdown produ cts of myelin basic protein (MBP) in the brain indicates the presence of in traumatic brain injury (TBI). Furthermore, t he concentration of these break down products can potentially be used to determine the severity of the injury. Thus, the role of proteins and peptides as potential biomarkers is important for study ing disease s and conditions such as TBI [2] The various aspects of protein structure and function can be assessed by analytical techniques such as 2 D gel e lectrophoresis, mass spectrometry ( MS ) and fluorescence [3] A common analytical approach involves extracting protein s from the tissue followed by separation by using chromatograph ic separation The fraction contai ning the protein of interest can then be purified by per forming gel electrophoresis. The bands of interest are excised digested with a protease and identified by peptide mass fingerprinting. Immunohistochemical staining can also be used to study a protei n in a tissue section in situ Alternatively, matrix assisted laser d esorption / i onization (MALDI) mass spectrometric imaging (MSI) can be used for the direct analysis of tissue to characterize or localiz e proteins and peptides [4] This technique avoids the homogenization and

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11 separation step s utilized in the aforementioned appro a ches thus preserving the spatial distribution of molecules within the tissue [5] Sample Preparation As with other analytical techniques s ample preparation for MSI is a critical step for generating high quality data and images Improper handling and storage of tissue can cause degradation or even delocalization of the analyte of interest. This section di scusses tissue preparation, storage, sectioning and matrix selection. Tissue Handling and Storage The tissue or organ is first surgically removed from the animal of study. Care should be taken to not only preserve the shape of the tissue but also prevent degradation. Immediately after removal, the tissue may be wrapped loosely in aluminum foil and flash frozen by gently submersing the wrapped tissue in liquid nitrogen for 30 to 60 seconds. Rapid submersion of the tissue is not recommended, as it may cause the tissue to crack. Direct submersion should also be avoided, as the tissue may adhere to the sides of the liquid nitrogen Dewar Also, the freshly excised tissue should not be placed immediately in plastic tubes. This may cause the tissue to take the sh ape of the plastic container or the tissue might also stick to the sides of the plastic tubes. After flash freezing, the whole tissue can be stored at 80 C for at least a year with little degra d ation [6] Tissue Sectioning and Mounting T issue sectioning is conducted in a cryostat chamber held between 5C to 25 C [7] Unlike the traditional tissue sectioning for histological staining, which u tilizes an embedding med ium such as optimal cutting temperature polymer (OCT) or agar tissue embedding is not recommended for MALDI mass spectrometry. The use of OCT has

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12 been reported to suppress analyte ion formation in MALDI MS studies [6] An alternative method to avoid the use of an embedding medium is to mount the tissue atop a few drop s of HPLC grade water on the cryostat mounting stage The low temperature of cryostat causes the water droplets to freeze holding the tissue in plac e on the mounting stage [8] After tissue mounting, f resh frozen tissues are sliced into thin sections (10 20 m) and thaw mounted on the MALDI plate. The thin sections can be transferred to a cold stainless stee l MALDI plate or conductive glass slide with t he aid of an artistic brush or forceps The tissue sections can also be transferred by placing the sample plate or the microscope slide on top of the section and allowin g the tissue to adhere [6] [7] The MALDI plate and the other tools used for sectioning like the artistic brush and forceps should be kept cold by placing them inside the cryostat. After the section is tr ansferred to the plate the tissue is gently heated by placing a finger on the opposite side of the plate. The mounted tissue should then be stored at 80C until further use to prevent it from enzymatic d egradation. Tissue Washing and Application of Matr ix A series of washing s teps may be beneficial for protein or peptide analysis before applying matrix since their detection is often prevented by large amounts of easily ionized lipids Larger molecules like proteins are not mobile enough to leave the tiss ue while washing. However, care must be taken for water soluble peptides while washing of the tissue section. Washing step s remove endogenous compounds such as lipids salts and improve signal quality [7] Wa shing can be performed by placing the tissue mounted MALDI plate in a petri dish, add ing the washing solution, swirl ing the dish for few seconds and discard ing the solution. Repeat the same with new washing solution.

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13 Selection of a proper matrix is a criti cal step in MALDI for obtaining high quality spectra. Selection depends on the type of analyte of interest. The matrix should absorb at the wavelength of the laser. Also, the matrix should co crystallize with analyte so that the analyte is desorbed in to g as phase upon ablation by the laser. The most commonly used MA LD I matrices are sinapinic acid cyano 4 hydroxycinnamic acid (CHCA) and 2, 5 hydroxybenzoic acid (DHB). SA is used for proteins with high molecular weight. CHCA and DHB are often used fo r smaller molecules like peptides and lipids. Careful deposition of matrix is critical to extract or desorb molecules efficiently and uniformly from the surface of the tissue. Uneven coating of the tissue with the matrix can cause variation in desorption o f the analyte from the tissue. Also, exc essive wetting of the tissue with the matrix can cause analyte migration. Therefore, there are several factors that must be considered to obtain uniform coating of the matrix. Matrix can be applied in a variety of wa ys. The common techniques used for coating matrix are, pneumatic spraying [9] i nkjet print ing [10] sublimation of matrix [11] and a coustic matrix deposition [12] Of all these techniques, pneumatic spraying is the most common technique. It can be performed by a nebulizer or even by a n artistic airbrush. These techniques can produce homogenous layer of small matrix crystals [7] Instrumentation Matrix Assisted Laser Desorption Ionization (MALDI) MALDI is a soft ionization technique used for small and large biomolecules The compounds to be analyzed are m ixed with a matrix solution The MALDI matrix is typically a small organic molecule that has strong absorption at the laser wavelength. The analyte/ matrix mixture is spotted atop a MALDI plate and dried before analysis forming matrix and analyte co crysta ls In case of tissue section the matrix is spray

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14 coated by using different spraying techniques as mentioned above in application of matrix section. Intense laser pulses induce rapid heating of the matrix and analyte co crystals causing sublimation Althoug h the MALDI ionization mechanism is not fully understood, singly charged analytes are generated from these co crystals which are then transferred to the mass spectrometer The matrix helps to minimize the sample damage from the laser by absorb ing most of the incident energy, increasing the efficiency of energy transfer from the laser to the analyt e. This technique is independent of the mass of the compound, which allows for the analysis of compounds with high molecular mass like proteins and peptides [13] [14] Time of Flight (TOF) Mass Analyzer The t ime of flight (TOF) mass analyzer operate s on the principle of measuring the time required for an ion to travel from the ion source to the detector. In linear time of flight (TOF) mass s pectrometry a s the ions are expelled from the source, each ion receive s the same kinetic energy S ince the ions have different m/z values, they have correspondingly different velocities The advantage of this type of ion separation is that, there is no theoretical upper mass limit [13] [14] The equation governing TOF ion separation is listed belo w (E quation 1 1 ) The m ass of an ion is m, the total charge (q) is equal to ze, and applied electrical potential is V s The electric potential energy is then converted to kinetic ener gy of an ion, which is represented by mv 2 /2. mv 2 /2 = zeV s (1 1) Equation 1 1 can be rearranged so that t he velocity of the ion leaving the source is given by:

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15 v = (2zeV s /m) (1 2) After acceleration, t he ion travels in a straight line to the detector The time t required for the ion to travel the distance L, before reaching t he detector is given by: t = L/v (1 3) Replacing v in equation 1 3 by equation 1 2 gives : t = L(m/2zeV s ) (1 4) Re arranging the terms: t 2 = (L 2 /2eV s ) m/z (1 5) Th u s equation 1 5 shows that m/z depends on the time required for the ion to travel the flig ht tube. A schematic detailing a linear time of flight detector is shown in figure 1 1. Since there is no theoretical upper mass limit for TOF analyzer, this advantage makes TOF mass analyzer suitable for operation with a MALDI ionization source [13] To improve the mass resolution of a TOF mass spectrometer, an electrostatic reflector, also known as a reflectron is used. The reflectron acts as a n ion mirror and is u s ed to reflect ions of the same m/z values t hat differ in kinetic energy The ion mirror utilizes electric field that opposes and has greater magnitude than the electric field in the ion acceleration region. The position of the mirror must be at an angle less than 180 to avoid reflection of ions ba ck into the source. The reflectron corrects the kinetic energy dispersion of the ions leaving the source with the same m/z ratio The ions with higher kinetic energy will penetrate the reflectron deepe r tha n ions with lower kinetic energy. This makes the f aster ions spend more time in the reflectron and reach the detector at the same time as slower ions with same m/z ratio [14] [13] A schematic detailing a reflectro n time of flight detector is shown in Figure 1 2.

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16 In MALDI imaging with TOF mass analyzer, ions are accelerated at a fixed potential travelling through a field free drift region called the flight tube, where these ions are separated in time based on their m/z ratio. Image analysis of molecules in tissue can be acquired using MALDI MS to determine spatial localization. The spectra from the tissue are recorded by moving the sample stage underneath a fixed laser beam. Each laser ablated spot, also called a pi xel give rise to a mass spectrum that correlates to an individual X, Y coordinate position on the tissue. Thus, each spot contain s dataset with different m/z values having its own intensity. The intensity is usually represented as a color scale. The intens ity of each m/z value can be arranged as a 2D ion density map. There are softwares available that can be used to generate images representing the localization and intensities of ions from a tissue section. TOF TOF Mass Spectrometer As in normal MALDI TOF o peration, extraction of ions is triggered by the laser pulse. The ions then pass through the field free region to allow the ions of various m/z values to separate sufficiently to be selec ted by the time gate to pass in to the collision cell. As the ions app roach the collision cell, it passes through a deceleration field where the kinetic energy of the ions is reduced to an operational value before entering the collision cell. As the ions travel through the collision cell, some are converted into product ions The high energy packet of ions extracted from the collision cell now enter a longer field free region to allow optimal separation of ion packets of various m/z values to produce a product ion spectrum. TOF TOF instrument is advantageous in proteomic as s equence information can be obtained in addition to molecular mass of proteolytic fragments for protein identification or analyses of amino acids.

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17 Peptide Fragmentation and Nomenclature Figure 1 3 illustrates a MS/MS fragmentation of a peptide with five a mino acids residue. Fragmentation of a peptide usually takes place at one of the bonds along the peptide backbone. On fragmentation six types of fragment ions can be generated that are termed as a, b, c x, y, and z type ions. The most common type of ions formed from the CID energy is the b and the y type ions. As shown in F igure 1 3, the b type ion extends from the amino terminus, also called as the N terminus, and the y type ion extends from the carboxyl terminus known as the C terminus [15] The number in the subscript denotes the number of amino acid remaining in the peptide fragment.

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18 Fig ure 1 1 Schmetic diagram of l inear time of flight ( TOF ) mass spectrometer Adapted from Watson, J.; Sparkman, D. Introduction t o Mass Spectrometry Instrumentation, Applications and Strategies for data Interpretation John Wiley and sons Ltd, 4 th Edition 2008 [14]

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19 Fig ure 1 2 Schmetic diagram of r eflectron time of flight ( TOF ) mas s spectrometer Adapted from Watson, J.; Sparkman, D., Introduction to Mass Spectrometry Instrumentation, Applications and Strategies for data Interpretation John Wiley and sons Ltd, 4 th Edition 2008 [14]

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20 Figure1 3. Nomenclature for peptide fragments generated by tandem mass spectrometry.

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21 CHAPTER 2 MALDI IMAGING OF MYE LIN BASIC PROTEIN IN TRAUMATIC BRAIN INJU RY Background Traumatic brain injury (TBI) causes a substantial number of deaths or permanent d isability in people. Recent data have shown that TBI is a serious health problem affecting over 1.7 million people every year in the United States [16] TBI may be caused by a sudden blow or j olt to the head or even a penetrat ion into the brain. The sources of TBI vary, including vehicular acci dents, violence, war and collision with moving or stationary objects. The symptoms of TBI can range from mild headaches t o severe problems like concussi ons, loss of memory, and even death Even though the rate of injury is high there are no effective treatments available for TBI [17] Additionally, i t is difficult to diagnose TBI using techniques like magnetic resonance imaging (MRI), computed tomography (CT) scanning or positron emission tomography (PET). Thus, a sensitive technique to determine specific biochemical markers following TBI may be beneficial for diagnosis [18] Neurotrauma following TBI promotes degradation of proteins in the brain. These degradation products which can either be smaller proteins or peptides may serve as biochemical markers for TBI that can be used to study the post injury mechan ism. An a xon is a long, slender projection of a neuron or nerve cell that serves to transmit signal s The axon which is located the white matter of the central nervous system is surrounded by a membrane called the myelin s heath. Myelin membranes protect and insulate neuron s and allow signal impulses to transmit efficiently along the neuron These membranes are composed of lipids and proteins layers, the main protein s being myelin basic protein (MBP), proteolipid protein and myelin

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22 oligodendrocyte glycopro tein Degradation of myelin proteins following TBI have been studied in demyelination diseases like multiple sclerosis. Among these myelin proteins, MBP is the most abundant protein and is presen t in various isoforms [19] [20] MBP has also been shown to degrade following a head injury. Hence, MBP may be considered as a biomarker for neurotrauma. MBP is a relatively high molecular weight protein Isoforms of MBP ranges fro m 8 kDa to 33 kDa. Proteins with such high molecular weight can be difficult to analyze by MS. Although there are mass spectrometers capable of detect ing proteins with high molecular weight, the sensitivity of such mass spectrometer s are significantly lowe r for high molecular weight proteins as compared to low molecular weight peptides. Therefore, there is a need to digest protein s with appropriate protease to obtain low molecular weight peptides that are representative of the original protein But m ass spe ctrometric analysis of such large proteins and peptides can be performed by using MALDI TOF instrument s since; these instruments have high mass range. The most commonly used protease is trypsin. Trypsin is a relatively stable protease, and cleaves at the c arboxyl terminal side of arginine and lysine residue For tryptic digestion of protein from the tissue section, it is necessary to extract the protein from the tissue before performing digestion. However, by performing extraction, the spatial resolution of the protein from the tissue is lost. Hence, there is a need to use an alternate technique to detect the protein from the tissue while preserving the spatial information The purpose of this work is to localize the intact MBP and study the breakdown produ cts of MBP in pathological samples from TBI by mass spectrometry.

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23 Experimental Materials and Chemicals MALDI matri ces sinapinic acid (SA) and cyano 4 hydroxycinnamic acid (CHCA) w ere purchased from Sigma Aldrich (St. Louis, MO) HPLC grade acetronitril e (ACN) and water were purchased from Honeywell B&J ( Muskegon, MI). HPLC grade m ethanol, acetic acid and trifluroacetic acid (TFA) were purchased from Fisher Scientific ( Fair Lawn, New Jersey). Chloroform was purchased from Acros organics (New Jersey) Tryp sin was purchased from Promega (Madison, WI). Trypsin solution was prepared to a final concentration of 12.5 ng/ L. SA and CHCA solution s w ere prepared to a final concentration of 10 mg/mL in 50 :50 ACN: 0.1% TFA in water 70% ethanol in water, 90:9:1 soluti on of ethanol:water:acetic acid and 100% chloroform were used as washing solutions to remove endogenous lipids and compounds from the tissue. MBP Standard MBP standard from human brain was purchased from Enzo Life Sciences (Plymouth, PA). The MBP standard solution of concentration 1mg/mL was prepared. The solution was mixed with SA solution (10 mg/mL) in the ratio of 1:1. 1 L of this mixture was spotted on a stainless steel MALDI plate. Analysis of MBP standard was performed in positive linear ionization mode. Samples Control human brain lysate, nave and injured ipsilateral cortex and ipsilateral hippocampus rat brain lysates were used for gel electrophoresis. Ipsilateral side is the same side of the injury whereas the opposite side is called contralater al side. B rain samples from adult male Sprague D aw ley rat s (200 250 g) were used The brains were removed; flash frozen and stored at 80 C until further use.

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24 Gel Electrophoresis The human and rat brain lysates were subjected to gel electrophoresis for th e separation of the protein s Following separation, the bands were excised and subjected to trypsin diges tion. The excised bands were washed with water a couple of time. The gel pieces were then wash ed 4 HCO 3 / ACN (50:50, v/v). The wash solution was then discarded. The gel pieces were dehydrated by adding 20 of ACN. Further, the dehydrated gel pieces were rehydrated with 15 of 12.5 ng/ trypsin solution and incubated at 4C for 30 min After incubation 20 uL of 50 mM NH 4 HCO 3 was added and kept for overnight incubat ion at 37C in a heating block Following overnight incubation the samples were centrifuged at 1500 rpm for 15 minutes. Supernatant was transferred to new tubes. 30 uL of 50% ACN / 50% water with 0.1% formic acid are added to the supernatant and centrifuged for 20 min at 1500 rpm. The supernatant was transferred to new tubes and subjected to evaporation using speed vacuum. The samples where then reconstituted with water with 0.1% formic acid The extracted peptide solution was mixed with MALDI matrix CHCA in the ratio of 1:1. 1 L of t his mixture was spotted on a stainless steel MALDI plate. Tissue Preparation Brain tissue was sectioned using a Microm HM 505 E cryostat (Waldorf, Germany) held at 25 C. The brain tissue was hel d on the cutting stage by dropping water around the tissue. The temperature of the cryostat causes the water to freeze holding the tissue in place. 10 m thick coronal sections were thaw mounted on either a stainless steel MALDI plate or ITO coate d glass slide. The brain sections were placed in a vacuum desiccator for 30 minutes to remove excess water. Further, a series of washes were performed to remove endogenous lipids

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25 and compounds fro m the tissue. The brain sections were washed twice with a solution of 7 0% ethanol in water for 30 seconds each. The sections were then washed with 100% chloroform for 30 seconds. Lastly, the sections were washed with a 90:9:1 solution of ethanol: water:acet ic acid (v:v:v) for 30 seconds. After the washing step the tissue sections wer e placed in the vacuum desiccator for 15 30 minutes to remove excess moisture. The most critical step in MALDI analysis is the unifo rm application of MALDI matrix. The tissue w as coated using glass Type A Meinhard N ebulizer (Golden, CO). Nitrogen was used as a nebulizing gas. SA (10 mg/ml) in 50 :50 ACN :0.1% TFA in water (v:v) was used as a MALDI matrix to study intact MBP from the brain tissue Instrumentation An AB Sciex MALDI TOF TOF 5800 mass spectrometer (Ontario, Canada) was used for the study of MBP The MALDI source consists of a diode pumped solid state Nd:YAG laser at 349 nm. The spot diameter of the laser is 100 m Analysis of tryptic peptides was performed using posi tive ion reflectron mode. Analysis of intact MBP from the brain section was performed using positive i on linear mode. In the linear mode (Figure 2 1) t he ions are accelerated in to the field free dr i ft region which is about 1.5 meters in length The ion o ptics steer and focus the ion beam towards the linear detector. I n the reflector mode (Figure 2 2) the ions are accelerated in to the field free dr i ft region which is about 3 meters in length. The ion optics steer and focus the ion beam towards the reflect or entrance. Further, the reflector focuses the ions of the same m/z to obtain better resolution and reflects the ion beam towards the reflector detector. For imaging, a raster step size of 1 2 0 m was used in continuous mode. The instrument range was scann ed from m/z 4 00 to m/z 2 ,000 for analysis of tryptic peptides in

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26 reflectron mode and from m/z 1000 to m/z 40,000 for analysis of intact protein in linear mode. Data Processing Pep tide sequences were evaluated with the University of California, San Francisco [21] Imaging data was acquired using For processing imaging data AB Tissue View software was used. Results and Discussion Figure 2 3 illustrates the MS spectrum of MBP standard. In this spectrum, displays the intact MBP. An 18 kDa isoform of MBP is observed as the dominant peak. Brain L ysate Analysis From the control human brain lysate well in the gel (Figure 2 4 ) a band near the 17 kDa marker band was excised. Also, a band from the ipsilateral hippocampus injured brain lysate well slightly below the 12 kDa marker band was excised. Thes e bands were subjected to trypsin digestion. Following tryp sin digestion the samples were analyzed by MS. Figure 2 6 and 2 7 shows the MS spectra of the tryptic peptides of MBP from control human brain lysate and the injured ipsilateral hippocampus (IH I) brain lysate respectively Most of the masses agree well with the tryptic peptide s predicted after performing in silico digestion using Protein Prospector (Figure 2 5 ) [21] Tryptic peptides of MBP appeared at m/z 69 8, 726, 1046, and 1460 from the control human brain lysate. Whereas tryptic peptides from the injured ipsilateral hippocampus (IH I) brain lysate appeared at m/z 726, 728, and 1046. MS 2 was performed on the se masses. In the MS 2 spectra of m/z 698 from cont rol human brain lysate (Figure 2 7 ) y 1 y 4 y 3 NH 3 y 4 NH 3

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27 y 5 NH 3 and b 2 ions are dominant. The MS 2 fragmentation data of m/z 728 from the injured ipsilateral hippocampus (IH I) brain lysate shows y 1 y 3 NH 3 y 4 NH 3 and b 2 as dominant ions (Figure 2 9) The dominant ions in the MS 2 spectra of m/z 726 from the control human brain lysate ( Figure 2 10 ) include b 2 b 3 and b 4 ions. The MS 2 spectrum of m/z 726 from the injured ipsilateral hippocampus (IH I) brain lysate shows y 1 y 2 b 2 b 3 and b 4 as domina nt ions (Figure 2 11). For the MS 2 fragmentation data of m/z 1046 (Figure 2 12 ) from the control human brain lysate y 4 and y 9 ions were dominant and t he MS 2 fragmentation data of m/z 1046 from the injured ipsilateral hippocampus (IH I) brain lysate shows y 1 y 4 and y 9 as dominant ions (Figure 2 1 3 ). The MS 2 spectra for m/z 1460 (Figure 2 1 4 ) observed only in the control human brain lysate, shows y 4 y 5 y 7 b 9 and b 10 ions were dominant. Direct Brain Analysis Coronal sections of the rat brain were selec ted for MSI analysis In the control brain section the two isoforms (14 kDa and 18 kDa) of MBP were identified The average mass spectrum of the coronal section from the control rat brain shows the two isoforms of MBP more distinctly ( F igure 2 1 5 A ). The 14 kDa isoform was observed to be more intense than the 18kDa isoform. The MSI images ( F igure 2 1 6 ) show t he distribution of these two isoforms of MBP in the corpus callosum and hippocampus of the brain The 14 kDa isoform sequence of MBP is composed of 12 8 amino acid s ( F igure 2 1 7 ), whereas 18 kDa isoform sequence of MBP is composed of 169 amino acids ( F igure 2 1 8 ). To study the effect of traumatic brain injury on the MBP, a controlled cortical impact (CCI) model was used. The average mass spectrum from t he coronal section of the TB I rat brain model (F igure 2 1 5 B ) show s a decrease in the signal intensity of 14

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28 kDa and 18 kDa isoform of MBP. The MSI images from the coronal section of the CCI brain ( F igure 2 19 ) also show the decrease in signal of the two i soforms of the MBP. The suspected breakdown products were observed in the TBI rat brain in the mass range of m/z 4980 5013 and m/z 9394 9537. Figure 2 2 0 shows t he MS I images of the suspected breakdown products T he distribution of these breakdown prod ucts were seen in the ipsilateral side, i.e. the side where the injury was induced. This indicate s there is breakdown or degradation of MBP after injury to the brain.

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29 Figure 2 1. Schmatic representation for linear mode operation on the AB SCIEX 5800 M ALDI TOF TOF instrument.

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30 Figure 2 2. Schmatic representation for reflector mode operation on the AB SCIEX 5800 MALDI TOF TOF instrument. The ion path for the product ions from MS 2 is shown in orange.

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31 Figure 2 3 MS spectrum of MBP standa r d. In this spectrum, the 18 kDa isoform of MBP form s dominant peak.

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32 Figure 2 4 Image of the 1 dimensional gel electrophoresis for the brain lysates from different regions and models ; human brain, ipsilateral cortex nave (IC N), ipsilateral hippocampus nave (IH N), ipsilateral cortex injured (IC I), ipsilateral hippocampus injured (IH I). A m olecular weight marker (MWM) was used as a reference.

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33 Figure 2 5 In silico digestion of MBP using Protein Prospector.

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34 Figure 2 6 MS spectrum of trypsin digestion of MBP band from control human brain lysate Figure 2 7 A) MS spectrum of trypsin digestion of MBP band from injured ipsilateral hippocampus (IH I) rat brain lysate. B) The zoom in spectrum displays the mass range m/z 700 750, to show m/z 726 and m/z 728 d istinctly.

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35 Figure 2 8 MS 2 spectrum of m/z 698 from control human brain lysate. Figure 2 9 MS 2 spectrum of m/z 72 8 from IH I rat brain lysate

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36 Figure 2 10 MS 2 spectrum of m/z 726 from control human brain lysate. Figure 2 1 1 MS 2 spectrum of m/z 726 from IH I rat brain lysate.

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37 Figure 2 12 MS 2 spectrum of m/z 1046 from control human brain lysate. Figure 2 1 3 MS 2 spectrum of m/z 1046 from IH I rat brain lysate.

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38 Figure 2 1 4 MS 2 spectrum of m/z 1460 from control human brain lysate.

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39 Fig ure 2 1 5 Average mass spectrum from the coronal section of the control rat brain.

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40 Figure 2 1 6 MSI images of coronal section of the control rat brain The two images illustrate the localization of different isoforms of MBP Image A shows the distribu tion of 14 kDa isoform of MBP and image B shows the distribution of 18 kDa isoform of MBP All intensities are normalized to the mean intensity of each pixel with baseline correction

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41 10 20 30 40 50 60 MASQKRPSQR HGSKYLATAS TMDHARHGFL PRHRDTGILD SIGRFFS GDR GAPKRGSGKD 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 SHTRTTHYGS LPQKSQRTQD ENPVVHFFKN IVTPRTPPPS QGKGRGLSLS RFSWGGRDSR SGSPMARR Figure 2 1 7 Rat MBP sequence for 14 kDa isoform. 10 20 30 40 50 60 MASQKRPSQR HGSKYLATAS TMDHARHGFL PRHRDTGILD SIGRFFSGDR GAP KRGSGKD 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 SHTRTTHYGS LPQKSQRTQD ENPVVHFFKN IVTPRTPPPS QGKGRGLSLS RFSWG AEGQK 130 140 150 160 PGFGYGGRAS DYKSAHKGFK GAYDAQGTLS KIFKLGGRDS RSGSPMARR Figure 2 1 8 Rat MBP sequence for 18 kDa isoform.

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42 Figure 2 19 MSI images of c oronal section of the TBI rat brain. Image A shows the distribution of 14 kDa isoform of MBP and image B shows the distribution of 18 kDa isoform of MBP All intensities are normalized to the mean intensity of each pixel with baseline correction.

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43 Figur e 2 2 0 MSI images of coronal section of the TBI rat brain showing the suspected breakdown products of MBP All intensities are normalized to the mean intensity of each pixel with baseline correction.

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44 CHAPTER 3 CONCLUSIONS AND FUTU RE WORK MALDI m ass spec trometric imaging provides excellent opportunities for identifying the molecular weight of an ion and its localization in the tissue section. By comparing the distribution of proteins between normal and injured brain, one can study the mechanism of injury. T his work illustrates the comparison between the normal and injured brai n tissue by MALDI imaging of MBP. The gel electrophoresis and MS data shows that in the IH I rat brain lysate, a band is observed a little below the 12 kDa marker band, which is absen t in the IH N rat brain lysate. This shows that there is breakdown of MBP following injury to the brain. Based on the gel electrophoresis results, localization of intact MBP in the control and injury rat brain was studied. A MALDI ion source in combinatio n with a TOF mass analyzer offers advantages for intact protein detection since there is no upper mass limit for TOF. MBP is present in different isoforms. In the control rat brain section, the 14 kDa and 18 kDa isoforms of MBP were identified as the most abundant peaks in the average mass spectrum of the tissue These isoforms were localized in the white matter, mainly in the corpus callosum and hippocampus region of the brain In the injured brain section the signal intensity for these two isoforms decr eases. This indicates there might be breakdown or degradation of MBP following injury to the brain. Substantial amount of work has been carried out to study TBI. Proteomics studies have been performed using serum and cerebrospinal fluid to study the break down products of different proteins following TBI. MBP i s not the only protein subject to breakdown following TBI. There are other proteins that might breakdown or degrade

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45 after a head injury. So far these studies have been performed by gel electrophoresis MALDI imaging would be an alternative method and probably the ideal technique to study the protein in TBI, as this technique can map the distribution of the analyte within the tissue. Future work is needed to study further the mechanism of degradation o r breakdown of this protein Furthermore, future work is needed to determine the other proteins involved in the breakdown process following TBI. This may include cutting out the individual regions of the brain, for instance the hippocampus or the corpus ca llosum, extracting the protein and performing digestion using an appropriate protease These digestion products can be used to further study the breakdown of the protein. T he ultimate purpose of this research is to demonstrate that protein breakdown produc ts can be used as injury biomarkers. Correlating the changes in the protein distribution in the normal and the injured tissue will help provide more understanding of the effect of injury on the proteins. This type of work can also be used to study the seve rity of the injury. Further more this can also help in the development of therapies for the patients with traumatic brain injury.

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46 LIST OF REFERENCES 1 Berg, J., Tymoczko, J. and Stryer, L. Biochemistry, 5th ed.; W. H. Freeman and Comp any:New York; 2002. 2 Mondello, S.; Jeromin, A.; Streeter, J.; Hayes, R.; Wang, K. Expert Rev. Mol. Diagn. 2011 11 (1) 65 78. 3 Wouters, F. S.; Verveer, P. J.; Bastiaens, P. I. H. Trends Cell Biol. 2001 11 203 211. 4 Caprioli, M.; Far mer, B.; Gile, J. Anal. Chem. 1997 69 4751 4760. 5 Seeley, E. H.; Caprioli, R. M. PNAS. 2008 105 18126 18131. 6 Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. J. Mass Spectrom. 2003 38 699 708. 7 Kaletas, B. K.; van der Wiel, I. M. ; Stauber, J.; Dekker, L. J.; Gzel, C.; Kros, J. M.; Luider, T. M.; Heeren, R. M. A. Proteomics. 2009 9 2622 2633. 8 Menger, R. F.; Stutts, W. L.; Anubukumar, D. S.; Bowden, J. A.; Ford, D. A.; Yost, R. A. Anal. Chem. 2012 84 (2) 1117 1125. 9 Garrett, T. J.; Prieto Conaway, M. C.; Kovtoun, V.; Bui, H.; Izgarian, N.; Stafford, G.; Yost, R. A. Int J Mass Spectrom. 2007 260 166 176. 10 Baluya, D. L.; Garrett, T. J.; Yost, R. A. Anal. Chem. 2007 79 6862 6867. 11 Hankin, J. A.; Barkley, R. M.; Murphy, R. C. J. Am. Soc. Mass Spectrom. 2007 18 1646 1652. 12 Aerni, H. R.; Cornett, D. S.; Caprioli, R. M. Anal. Chem. 2006 78 827 834. 13 de Hoffmann, E. and Stroobant, V. Mass Spectrometry: Principles and Applications, Thir d ed.; John Wiley and Sons, Ltd. 2009. 14 Watson, T. J. and Sparkman, D. Introduction to Mass Spectrometry Instrumentation, Applications and Strategies for Data Interpretation, 4th ed.; John Wiley & Sons, Ltd. 2008. 15 Paizs, B.; Suhai, S Mass Spectrom. Rev. 2005 24 508 548. 16 Centers for Disease Control and Prevention ( http://www.cdc.gov/ ) (Accessed February 2012).

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47 17 Kobeissy, F. H.; Sadasivan, S.; Oli, M. W.; Robinson, G.; Larner, S. F. ; Zhang, Z.; Hayes, R. L.; Wang, K. K. W. PROTEOMICS Clin Appl. 2008 2 1467 1483. 18 Kobeissy, F. H.; Ottens, A. K.; Zhang, Z.; Liu, M. C.; Denslow, N. D.; Dave, J. R.; Tortella, F. C.; Hayes, R. L.; Wang, K. K. W. Mol Cell Proteomics. October 2 006 5 1887 1898. 19 Liu, M. C.; Akle, V.; Zheng, W.; Kitlen, J.; O'Steen, B.; Larner, S. F.; Dave, J. R.; Tortella, F. C.; Hayes, R. L.; Wang, K. K. W. J.Neurochem. 2006 98 700 712. 20 Ottens, A. K.; Golden, E. C.; Bustamante, L.; Hayes, R. L.; Denslow, N. D.; Wang, K. K. W. J.Neurochem. 2008 104 1404 1414. 21 Protein Prospector ( http://prospector.ucsf.edu/ ) (Accessed January 2012).

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48 BIOGRAPHICAL SKETCH Manasi Mangaonkar was born in October 1983 as a second child to Anuradha and Dil i p Mangaonkar in Mumbai, India. She did her schooling in Mumbai. She received a BS in life sciences in 2004 and a MS in bioanalytical sciences in 2006 from the University of Mumbai. During her MS studies she was introduced to mass spectrometry. After finishing her MS, she joined SITEC Labs, a bioanalytical division of Cipla Pharmaceuticals as an analyst where she worked for two and half years. She married Naren Kamat in December 2008 and moved to Florida S he then decided to continue with her studi es at the University of Florida (UF) and joined the ch emistry department as a graduate student. At UF, Manasi did her research in the field of mass spectrometric imaging to study the effect of traumatic brain in jury on the proteins in the brain, under the direction of Dr. David Powell and Dr. Richard Yost. In the summer of 2012, Manasi graduated with a Master of Science in analytical chemistry.