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PROTEOMIC ANALYSIS of HUMAN URINARY EXOSOMES. Patricia

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PROTEOMIC ANALYSIS of HUMAN URINARY EXOSOMES. Patricia ABSTRACT Title of Document: PROTEOMIC ANALYSIS OF HUMAN URINARY EXOSOMES. Patricia Amalia Gonzales Mancilla, Ph.D., 2009 Directed By: Associate Professor Nam Sun Wang, Department of Chemical and Biomolecular Engineering Exosomes originate as the internal vesicles of multivesicular bodies (MVBs) in cells. These small vesicles (40-100 nm) have been shown to be secreted by most cell types throughout the body. In the kidney, urinary exosomes are released to the urine by fusion of the outer membrane of the MVBs with the apical plasma membrane of renal tubular epithelia. Exosomes contain apical membrane and cytosolic proteins and can be isolated using differential centrifugation. The analysis of urinary exosomes provides a non- invasive means of acquiring information about the physiological or pathophysiological state of renal cells. The overall objective of this research was to develop methods and knowledge infrastructure for urinary proteomics. We proposed to conduct a proteomic analysis of human urinary exosomes. The first objective was to profile the proteome of human urinary exosomes using liquid chromatography-tandem spectrometry (LC- MS/MS) and specialized software for identification of peptide sequences from fragmentation spectra. We unambiguously identified 1132 proteins. In addition, the phosphoproteome of human urinary exosomes was profiled using the neutral loss scanning acquisition mode of LC-MS/MS. The phosphoproteomic profiling identified 19 phosphorylation sites corresponding to 14 phosphoproteins. The second objective was to analyze urinary exosomes samples isolated from patients with genetic mutations. Polyclonal antibodies were generated to recognize epitopes on the gene products of these genetic mutations, NKCC2 and MRP4. The potential usefulness of urinary exosome analysis was demonstrated using the well-defined renal tubulopathy, Bartter syndrome type I and using the single nucleotide polymorphism in the ABCC4 gene. The third objective was to study the normal variability between proteomes of female and male urinary exosomes, and to implement a normalization method to analyze urinary exosome samples. Only 19 proteins had a 2-fold change representing 4.9% of the total number of proteins identified which shows that there is high concordance between proteomes of urinary exosomes isolated from males and females. The normalization method, timed urine collection did not correlate as expected with the intensity signal of MVB markers, TSG101 and Alix. This research shows that the proteomic analysis of human urinary exosomes can be the basis for future biomarker studies as well as physiological studies. PROTEOMIC ANALYSIS OF HUMAN URINARY EXOSOMES. By Patricia Amalia Gonzales Mancilla Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2009 Advisory Committee: Associate Professor Nam Sun Wang, Chair Dr. Mark A. Knepper, Special Graduate Faculty Member Associate Professor Sheryl Ehrman Professor William E. Bentley Professor Catherine Fenselau © Copyright by Patricia Amalia Gonzales Mancilla 2009 Dedication I dedicate this work to my husband, my daughter, my parents and my brother. ii Acknowledgements I would like to thank my advisors: Dr. Nam Sun Wang, Associate Professor in the Department of Chemical and Biomolecular Engineering at the University of Maryland- College Park and Dr. Mark A. Knepper, Chief of the Laboratory of Kidney and Electrolyte Metabolism (LKEM) at the National Heart, Lung, Blood Institute (NHLBI) of the National Institutes of Health (NIH) for their continued guidance and support during my graduate career. The research experience that I gained as a graduate student is invaluable. Also, I would like to thank my committee members for their advice and support in the completion of this work. I am very grateful to Dr. Trairak Pisitkun for his endless help and mentoring while conducting my dissertation research. Thanks to Dr. Robert E. Kleta for his continued collaboration to applying urinary exosomes analysis to clinical medicine. Thanks to Dr. Robert A. Star and Dr. Hua Zhou for their valuable collaboration in biomarker discovery pertaining to urinary proteomics. Thanks to Dr. Jeffrey Kopp for his collaboration in the gender proteomics study. To all the members of the Laboratory of Kidney and Electrolyte Metabolism, thank you for all your support. A special thanks to the Clark School’s Future Faculty Program for providing additional training that will serve me in the future. To all my friends in college and graduate school, I will treasure your support and friendship forever. To my parents and brother, thank you for your love, trust, understanding, guidance and continuous support in all my life endeavors. To my husband, thank you for your unconditional love, friendship, and support. To my daughter, thank you for inspiring me. iii Table of Contents Dedication ........................................................................................................................... ii Acknowledgements ............................................................................................................ iii Table of Contents ............................................................................................................... iv List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii 1 Introduction ................................................................................................................. 1 2 Background ................................................................................................................. 5 2.1 Human urinary exosomes ......................................................................................5 2.1.1 Introduction ....................................................................................................5 2.1.2 Urinary exosome biogenesis ..........................................................................7 2.1.3 Summary ........................................................................................................8 2.2 Proteomics .............................................................................................................9 2.2.1 Introduction ....................................................................................................9 2.2.2 Tandem Mass Spectrometry ..........................................................................9 2.2.3 Analysis of tandem mass spectrometry data ................................................11 2.2.4 Summary ......................................................................................................12 2.3 Development of urinary clinical proteomics .......................................................12 3 Large Scale Proteomic Analysis of Human Urinary Exosomes * ............................ 14 3.1 Introduction .........................................................................................................14 3.2 Methods ...............................................................................................................15 3.2.1 Large-scale proteomic analysis ....................................................................15 3.2.1.1 Urinary Exosome Isolation .................................................................. 15 3.2.1.2 In-Gel Trypsin Digestion ..................................................................... 17 3.2.1.3 Nanospray LC-MS/MS ........................................................................ 18 3.2.1.4 Analysis of Data .................................................................................. 18 3.2.1.5 Target-Decoy ....................................................................................... 18 3.2.1.6 InsPecT ................................................................................................ 19 3.2.1.7 Minimizing False-Discovery Peptide Identifications .......................... 20 3.2.1.8 Elimination of Ambiguous Protein Identifications .............................. 20 3.2.2 Phosphoproteomic Analysis .........................................................................22 3.2.2.1 Phosphopeptide Enrichment ................................................................ 22 3.2.2.2 Analysis of Phosphopeptide Data Sets ................................................ 23 3.3 Results .................................................................................................................24 3.3.1 Large-Scale Proteomic Profiling of Human Urinary Exosomes ..................24 3.3.2 Phosphoproteomic Analysis of Human Urinary Exosomes .........................25 3.4 Discussion ...........................................................................................................28 3.4.1 Large-Scale Proteomic Profiling of Human Urinary Exosomes ..................28 3.4.2 Relevance to Renal Biology .........................................................................29 3.4.3 Phosphoproteomic Analysis of Human Urinary Exosomes .........................30 3.5 Conclusion ...........................................................................................................30 4 Application of urinary exosomes analysis using polyclonal antibodies ................... 43 4.1 Introduction
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