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HIGH MASS ACCURACY COUPLED TO SPATIALLY-DIRECTED PROTEOMICS FOR
IMPROVED PROTEIN IDENTIFICATIONS IN IMAGING MASS SPECTROMETRY
EXPERIMENTS

By
David Geoffrey Rizzo
Dissertation
Submitted to the Faculty of the
Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY in

Chemistry August, 2016
Nashville, Tennessee

Approved:
Richard M. Caprioli, Ph.D.
Kevin L. Schey, Ph.D. John A. McLean, Ph.D. Michael P. Stone, Ph.D.

i

Copyright © 2016 by David Geoffrey Rizzo
All Rights Reserved

ii

This work is dedicated to my family and friends, who have shown nothing but support for me in

all of life’s endeavors.

iii

ACKNOWLEDGEMENTS

“As we express our gratitude, we must never forget that the highest appreciation is not to utter words, but to live by them.”

- John F. Kennedy –

There are many people I must thank for showing kindness, encouragement, and support for me during my tenure as a graduate student. First and foremost, I would like to thank my research advisor, Richard Caprioli, for providing both ample resources and guidance that allowed me to grow as a scientist. Our discussions about my research and science in general have helped me become a much more focused and discerning analytical chemist. I must also thank my Ph.D. committee members, Drs. Kevin Schey, John McLean, and Michael Stone, who have brought valuable insight into my research and provided direction along the way. My undergraduate advisor, Dr. Facundo Fernández, encouraged me to begin research in his lab and introduced me to the world of mass spectrometry. It was his motivation that drove me to pursue my Ph.D.
Every member of the Mass Spectrometry Research Center, both past and present, has guided me in some form or fashion. I would not be where I am today without the support of my “lab family”, both on a personal and scientific level. Maureen Casey was always there to

brighten up the lab with humor and support. I will certainly miss going to see “Mama Maureen”

and have her fix any problem almost immediately. Dr. Peggi Angel advised me as a rotation student when I first came to Vanderbilt and I strived to emulate her passion for science. Amanda Hachey, Salisha Hill, and Dr. Kristie Rose in the Proteomics Core could not have been more accommodating as I plunged into the vast field of proteomics. They were always there to share their wisdom, helping me learn new techniques that will be invaluable in my career. Drs. Megan

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Gessel, Joshua Nicklay, Glenn Harris, Jeffrey Spraggins, and Boone Prentice have all been vital to my development as a scientist, acting as soundboards for ideas and encouraging me to enjoy successes and learn from failures. Whenever I needed to vent about science or life, they were there to listen. The fellow graduate students in lab who have seen me through this journey have left a lasting impression on me as well. They have always been there to lend a hand and I will truly miss seeing them in lab every day.
None of this would be possible without support from my family and friends. My mother, Robin, and father, Joe, encouraged me to pursue my love of science from an early age, even if that meant driving to enrichment programs early Saturday mornings. They taught me how to be a hard worker and independent person, but to never forget that no man is an island. Success and humility should never be mutually exclusive. My brother, Joey, and my sister, Meghan, continue to encourage me every day in everything that I do. Meghan has listened to the recap of my current state of life at day’s end, and for that, I am eternally grateful. I cannot forget Rebecca Rosenberg, who has been a part of our family since the first grade. We have grown up together and will always be there to ground each other, no matter how far apart we are. My dogs, Marco and Polo, are the first things I see when I wake up and the last things I see when I go to bed.

They’ve made the roughest days better, always showing excitement and affection when I get

home (no matter how late that may be). Lastly, I would like to thank my entire extended family, including all of my aunts, uncles, and cousins. Though my grandparents could not be here to see this achievement, I know they would all be proud of such an accomplishment.
I must also acknowledge the funding support of grants from the NIH/NIGMS (5P41
GM103391-05 and 5R01 GM058008), the NIH Shared Instrumentation Grant Program (1S10OD012359-01), and a fellowship from Aegis Sciences Corporation.

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TABLE OF CONTENTS
Page
DEDICATION............................................................................................................................... iii ACKNOWLEDGEMENTS........................................................................................................... iv TECHNICAL ABBREVIATIONS................................................................................................ ix LIST OF TABLES......................................................................................................................... xi LIST OF FIGURES ..................................................................................................................... xiii Chapter I. INTRODUCTION & RESEARCH OBJECTIVES...................................................................1
Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry ..........................................1 Imaging Mass Spectrometry.....................................................................................................3 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry...........................................5 Spatially-Directed Tissue Collection Methods.........................................................................9 Traditional Approaches to Protein Identification in IMS Experiments..................................11 Summary and Research Objectives........................................................................................14

II. MALDI FTICR IMS OF INTACT PROTEINS: USING MASS ACCURACY
TO LINK PROTEIN IMAGES WITH PROTEOMICS DATA..............................................16

Introduction ............................................................................................................................16 Methodologies for MALDI FTICR IMS and Identification of Intact Proteins......................17
Tissue Preparation..............................................................................................................17 Matrix Application.............................................................................................................17 MALDI FTICR IMS..........................................................................................................18 Histology............................................................................................................................18 Protein Purification for Identifications ..............................................................................19 LC-Coupled Tandem Mass Spectrometry (ETD)..............................................................19 Data Analysis.....................................................................................................................20
MALDI FTICR IMS of Rat Brain as a Model System for Protein Imaging..........................21 Observed Protein Expression in clear cell Renal Cell Carcinoma (ccRCC)..........................36 Conclusions ............................................................................................................................39

III. SPATIALLY-TARGETED EXTRACTIONS TO IMPROVE PEPTIDE
IDENTIFICATIONS FROM MALDI FTICR IMS EXPERIMENTS....................................40

Introduction ............................................................................................................................40

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Methodologies for Trypsin Application, in situ Digestion, Incubation and MALDI IMS for Peptides.......................................................................................................41
Tissue Preparation..............................................................................................................41 Trypsin Digestion...............................................................................................................42 Matrix Application.............................................................................................................42 MALDI FTICR IMS .........................................................................................................43 Peptide Extraction from Tissue..........................................................................................43 LC-Coupled Tandem Mass Spectrometry (HCD) .............................................................43 Data Analysis.....................................................................................................................44
Optimizing Incubation Conditions to Maximize Digestion and Minimize Delocalization.........................................................................................................................44 MALDI FTICR IMS and Localized Peptide Analysis of Biological Samples ......................48 Conclusions ............................................................................................................................57

IV. ADVANCED HYDROGEL TECHNOLOGIES FOR ENHANCED ON-TISSUE
PROTEIN DIGESTION AND IMPROVED SPATIAL LOCALIZATION...........................58

Introduction ............................................................................................................................58 Methodologies for Hydrogel Comparisons & Analysis .........................................................60
Tissue Preparation and In-Solution Digests.......................................................................60 Hydrogel Workflow...........................................................................................................61 LC-Coupled Tandem Mass Spectrometry (HCD) .............................................................62 Data Analysis.....................................................................................................................63
Optimizing Trypsin Concentration for Efficient On-Tissue Digestion..................................64 Increasing Polyacrylamide Percentages for Improved Hydrogel Rigidity.............................69 Fabricating Smaller Hydrogels for More Localized Analyte Extraction ...............................80 Comparison of Sample Collection Methods...........................................................................85 Differential Proteomic Analysis of Rat Cerebellum Substructures........................................88 Conclusions ............................................................................................................................91

V. CONCLUSIONS & PERSPECTIVES ...................................................................................93
MALDI FTICR IMS of Intact Proteins..................................................................................93 Spatially-Targeted Extractions Coupled to MALDI FTICR IMS Experiments.....................94 Advanced Hydrogel Technologies .........................................................................................95 Future Research Directions ....................................................................................................96 Conclusions ............................................................................................................................97

Appendix A. Protein Identifications Unique to the "White Matter" Hydrogel............................................99 B. Protein Identifications Unique to the "Molecular Layer" Hydrogel ....................................107

C. Protein Identifications Common to both the “White Matter” and "Molecular Layer"

Hydrogels..............................................................................................................................112

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References....................................................................................................................................132 Curriculum Vitae .........................................................................................................................144

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TECHNICAL ABBREVIATIONS
2-NPG: 2-nitrophloroglucinol 9AA: 9-aminoacridine ANOVA: analysis of variance ACN: acetonitrile ccRCC: clear cell renal cell carcinoma CHCA: α-cyano-4-hydroxycinnamic acid CID: collision induced dissociation CMBT: 5-chloro-2-mercaptobenzothiazole CT: computed tomography Da: Dalton DAN: 1,5-Diaminonapthalene df: degrees of freedom DHA: 2,5-dihydroxyacetophenone DHB: 2,5-dihydroxybenzoic acid ESI: electrospray ionization ETD: electron-transfer dissociation FID: free induction decay FTICR: Fourier transform ion cyclotron resonance H&E: hemotoxylin and eosin HPLC: high-performance liquid chromatography IHC: immunohistochemistry

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IMS: imaging mass spectrometry ITO: indium-tin-oxide LC: liquid chromatographic LCM: laser capture microdissection MALDI: matrix-assisted laser desorption/ionization MRI: magnetic resonance imaging MS: mass spectrometry MS/MS: tandem MS Mr: molecular mass m/z: mass-to-charge ratio Nd:YAG: neodymium-doped yttrium aluminum garnet Nd:YLF: neodymium-doped yttrium lithium garnet ppm: parts per million RF: radio frequency RP: resolving power SA: sinapinic acid S/N: signal-to-noise ratio THAP: 2′,4′,6′-trihydroxyacetophenone TOF: time-of-flight TOF/TOF: time-of-flight/time-of-flight UV: ultraviolet

x

LIST OF TABLES
Table 3.1. Protein Identifications from Rat Brain Regions Exclusively Observed by
Page
Microextraction Analysis...............................................................................................................46 3.2. Protein Identifications from Resected Human ccRCC Tumor Regions Exclusively Observed by Microextraction Analysis......................................................................48

4.1. Average Numbers of Identified Trypsin Autolytic Peptides from Hydrogels with Different Trypsin Concentrations ..........................................................................................59

4.2. Analysis of Variance (ANOVA) for Numbers of Identified Trypsin Autolytic Peptides from Hydrogels with Different Trypsin Concentrations .................................................60

4.3. Tukey’s Multiple Comparison Test for Differences in Numbers of Identified Trypsin Autolytic Peptides from Hydrogels with Different Trypsin Concentrations....................60

4.4. Average Numbers of Protein Identifications from Hydrogels with Different Trypsin Concentrations..................................................................................................................61

4.5. Analysis of Variance (ANOVA) for Numbers of Protein Identifications from Hydrogels with Different Trypsin Concentrations ........................................................................61

4.6. Tukey’s Multiple Comparison Test for Differences in Numbers of Protein Identifications from Hydrogels with Different Trypsin Concentrations........................................62

4.7. Gel Formulation Protocols for Each Polyacrylamide Percentage...........................................63 4.8. Average Numbers of Protein Identifications from Hydrogels with Different Polyacrylamide Percentages ..........................................................................................................64

4.9. Analysis of Variance (ANOVA) for Numbers of Protein Identifications from Hydrogels with Different Polyacrylamide Percentages.................................................................64

4.10. Tukey’s Multiple Comparison Test for Differences in Numbers of Protein Identifications from Hydrogels with Different Polyacrylamide Percentages................................64

4.11. Molecular Weight Bins of Protein Identifications from 7.5% and 18% Polyacrylamide Hydrogels and the Uniprot Rat Proteome............................................................66

4.12. Average Numbers of Protein Identifications from Control (Blank) Hydrogels ...................67

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4.13. Average Numbers of Protein Identifications from the Analysis of a Technical Replicate Experiment Repeated Three Times...............................................................69

4.14. Average Diameter Measurements of Hydrogels Fabricated with Different Diameter Biopsy Punches..............................................................................................................70

4.15. Average Numbers of Protein Identifications from Hydrogels Fabricated with Different Diameter Biopsy Punches ......................................................................................72

4.16. Analysis of Variance (ANOVA) for Numbers of Protein Identifications from Hydrogels Fabricated with Different Diameter Biopsy Punches...................................................72

4.17. Tukey’s Multiple Comparison Test for Differences in Numbers of Protein Identifications from Hydrogels Fabricated with Different Diameter Biopsy Punches..................72

4.18. Average Numbers of Protein Identifications from Different Sample Collection Methods..........................................................................................................................................75

4.19. Analysis of Variance (ANOVA) for Numbers of Protein Identifications from Different Sample Collection Methods ...........................................................................................75

4.20. Tukey’s Multiple Comparison Test for Differences in Numbers of Protein Identifications from Different Sample Collection Methods ..........................................................75

4.21. The 10 Highest Ranked Hydrogel-Mediated Protein Identifications Unique to the White Matter or Molecular Layer of the Rat Cerebellum ...................................................78

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LIST OF FIGURES

  • Figure
  • Page

1.1. Structures of Common MALDI Matrices.................................................................................2 1.2. Schematic Outline of a Typical IMS Workflow for Fresh Frozen Tissue Samples .................4 1.3. Defining Mass Resolution and Mass-Measurement Precision .................................................5 1.4. Fourier Transform Ion Cyclotron Resonance (FTICR) MS Signal Detection Process ............8 1.5. Implementation of Laser Capture Microdissection.................................................................10

1.6. Schematic Overview of ‘Bottom-Up’ and ‘Top-Down’ Approaches Employed

for Tandem Mass Spectrometry-Based Protein Identification and Characterization. ...................12 2.1. General Sample Preparation Workflow for MALDI FTICR Protein IMS and Subsequent Identification Strategies Using ETD Fragmentation..................................................20

2.2. MALDI FTICR IMS Average Spectra of Intact Proteins from Transversally Sectioned Rat Brain Tissue............................................................................................................22

2.3. MALDI FTICR IMS Data Collected from Transversally Sectioned Rat Brain Tissue.............................................................................................................................................24

2.4. Selected Ion Images of Identified Intact Proteins from Rat Brain Tissue Collected Using MALDI FTICR IMS...........................................................................................26

2.5. High Mass Resolution Top-Down ETD for the [Mꢀ+ꢀ8H]8+ Charge State of Thymosin β4 ..................................................................................................................................29

2.6. ETD LC-MS/MS Data for N-Terminally Acetylated Thymosin β10 ......................................30 2.7. ETD LC-MS/MS Data for ATP Synthase Subunit Epsilon....................................................31 2.8. ETD LC-MS/MS Data for Histone H4 (N-Acetylated, K21 Dimethylated) ..........................32 2.9. ETD LC-MS/MS Data for Histone H4 (N-Acetylated, K17 Acetylated, K21 Dimethylated).........................................................................................................................33

2.10. Selected Ion Images of Identified Intact Proteins from Human clear cell Renal Cell Carcinoma Collected Using MALDI FTICR IMS.......................................................35

xiii

3.1. General Sample Preparation Workflow for MALDI FTICR Peptide IMS and Subsequent Identification Strategies Using CID/HCD Fragmentation...................................42

3.2. Utilizing MALDI IMS to Aid in the Assessment of Enzymatic Incubation Conditions......................................................................................................................................44

3.3. LC-MS/MS Analysis of Rat Brain Regions Sampled by Microextraction and Tissue Homogenization .................................................................................................................45

3.4. LC-MS/MS Analysis of Resected Human ccRCC Tumor Regions Sampled by Microextraction and Tissue Homogenization...........................................................................47

3.5. MALDI FTICR IMS of Rat Brain Peptides Identified By Localized Extraction Methods..........................................................................................................................................50

4.1. General Hydrogel Fabrication, Usage, and Extraction Workflow..........................................58 4.2. Average Numbers of Identified Trypsin Autolytic Peptides from Hydrogels with Different Trypsin Concentrations ..........................................................................................59

4.3. Average Numbers of Protein Identifications from Hydrogels with Different Trypsin Concentrations..................................................................................................................62

4.4. Average Numbers of Protein Identifications from Hydrogels with Different Polyacrylamide Percentages ..........................................................................................................65

4.5. Molecular Weight Distributions of Protein Identifications from 7.5% and 18% Polyacrylamide Hydrogels and the Uniprot Rat Proteome............................................................66

4.6. Average Numbers of Protein Identifications from Control (Blank) Hydrogels .....................67 4.7. Average Numbers of Protein Identifications from the Analysis of a Technical Replicate Experiment Repeated Three Times................................................................................69

4.8. Differential Comparison of Protein Identifications from a Technical Replicate Experiment Repeated Three Times................................................................................................69

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    Génomique des populations et association génotype- phénotype des écotypes de touladi du lac Supérieur Mémoire Alysse Perreault-Payette Maîtrise en biologie Maître ès sciences (M. Sc.) Québec, Canada © Alysse Perreault-Payette, 2016 Génomique des populations et association génotype- phénotype des écotypes de touladi du lac Supérieur Mémoire Alysse Perreault-Payette Sous la direction de : Louis Bernatchez, directeur de recherche Pascal Sirois, codirecteur de recherche Résumé L’apparition et le maintien d’écotypes adaptés à différentes niches écologiques, en situation de sympatrie, est régit par une multitude de facteurs. Ceux-ci sont essentiels pour la compréhension des processus évolutifs impliqués mais aussi pour la gestion et la conservation des populations en question. Le touladi (Salvelinus namaycush) est un salmonidé reconnu pour la présence d’écotypes liée à l’utilisation des ressources et de l’habitat à travers l’Amérique du Nord. Un total de quatre écotypes a été décrit vivant dans le lac Supérieur, se différenciant par l’habitat utilisé, l’alimentation, la morphologie ainsi que l’ostéologie. L’objectif principal de la présente étude était de quantifier l’étendue de la différentiation génétique entre les différents sites d’échantillonnage ainsi qu’entre les différents écotypes. Un second objectif était d’identifier des marqueurs potentiellement sous sélection entre les différents écotypes reflétant de possibles adaptations locales. Pour ce faire, un total de 486 individus, représentant les quatre écotypes pour chacun des quatre sites d’échantillonnages, a été génotypé à 6822 SNPs (polymorphisme de nucléotide simple). De plus, des analyses morphométriques ont été effectuées afin de caractériser l’ampleur de la divergence morphologique entre les écotypes à chacun des sites.
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation

    Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation

    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
  • Supplementary Information.Pdf

    Supplementary Information.Pdf

    Supplementary Information Whole transcriptome profiling reveals major cell types in the cellular immune response against acute and chronic active Epstein‐Barr virus infection Huaqing Zhong1, Xinran Hu2, Andrew B. Janowski2, Gregory A. Storch2, Liyun Su1, Lingfeng Cao1, Jinsheng Yu3, and Jin Xu1 Department of Clinical Laboratory1, Children's Hospital of Fudan University, Minhang District, Shanghai 201102, China; Departments of Pediatrics2 and Genetics3, Washington University School of Medicine, Saint Louis, Missouri 63110, United States. Supplementary information includes the following: 1. Supplementary Figure S1: Fold‐change and correlation data for hyperactive and hypoactive genes. 2. Supplementary Table S1: Clinical data and EBV lab results for 110 study subjects. 3. Supplementary Table S2: Differentially expressed genes between AIM vs. Healthy controls. 4. Supplementary Table S3: Differentially expressed genes between CAEBV vs. Healthy controls. 5. Supplementary Table S4: Fold‐change data for 303 immune mediators. 6. Supplementary Table S5: Primers used in qPCR assays. Supplementary Figure S1. Fold‐change (a) and Pearson correlation data (b) for 10 cell markers and 61 hypoactive and hyperactive genes identified in subjects with acute EBV infection (AIM) in the primary cohort. Note: 23 up‐regulated hyperactive genes were highly correlated positively with cytotoxic T cell (Tc) marker CD8A and NK cell marker CD94 (KLRD1), and 38 down‐regulated hypoactive genes were highly correlated positively with B cell, conventional dendritic cell