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Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion

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Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Summer 2009 Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion Todd M. Greco University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, and the Molecular and Cellular Neuroscience Commons Recommended Citation Greco, Todd M., "Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion" (2009). Publicly Accessible Penn Dissertations. 22. https://repository.upenn.edu/edissertations/22 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/22 For more information, please contact [email protected]. Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion Abstract The ability of astrocytes to secrete proteins subserves many of its known function, such as synapse formation during development and extracellular matrix remodeling after cellular injury. Protein secretion may also play an important, but less clear, role in the propagation of inflammatory responses and neurodegenerative disease pathogenesis. While potential astrocyte-secreted proteins may number in the thousands, known astrocyte-secreted proteins are less than 100. To address this fundamental deficiency, mass spectrometry-based proteomics and bioinformatic tools were utilized for global discovery, comparison, and quantification of astrocyte-secreted proteins. A primary mouse astrocyte cell culture model was used to generate a collection of astrocyte-secreted proteins termed the astrocyte secretome. A multidimensional protein and peptide separation approach paired with mass spectrometric analysis interrogated the astrocyte secretome under control and cytokine-exposed conditions, identifying cytokine- induced secreted proteins, while extending the depth of known astrocyte-secreted proteins to 169. Several of these proteins were likely secreted by non-conventional mechanisms. These non-conventional mechanisms were explored further using stable isotope labeling by amino acids in cell culture, revealing 12 putative non-conventionally secreted proteins. These qualitative and quantitative mass spectrometry approaches are broadly applicable for the study of cellular secretomes as well as for extension to in vivo secretomes. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Neuroscience First Advisor Harry Ischiropoulos Keywords Mass Spectrometry, Proteomics, Astrocyte, Protein Secretion, Nitric Oxide, S-nitrosylation Subject Categories Cell Biology | Molecular and Cellular Neuroscience This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/22 MASSSPECTROMETRY.BASED PROTEOMICS REVEALS DISTINCTMECHANISMS OF ASTROCYTEPROTEIN SECRETION Todd MichaelGreco A DISSERTATION 1n Neuroscience Presentedto the Facultiesof the lJniversityof Pennsylvania in PartialFulfillment of the Requirementslbr the Degreeof Doctorof Philosophy 2009 Supervisorof Dissertation:Harry Ischirdpoulos. Ph.D. 'I!i" i\\\ \\ lv\.v"1 GraduateGroup Cl-rairperson: Michael P. Nusbaum.Ph.D. DissertationComrnittee Tom Parsons.Ph.D. MatthewDalva" Ph.D. IanBlair. Ph.D. StevenSeeholzer. Ph.D. Mass Spectrometry-based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion COPYRIGHT 2009 Todd Michael Greco To My Parents who have made my academic endeavours and life pursuits become reality with their unending support iii ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Harry Ischiropoulos, for the opportunity to conduct my doctoral work in his lab. Without his support as a mentor, colleague, and friend, none of this work would be possible. His passion for scientific inquiry and ability to teach the scientific method by example has made a lasting impression on my graduate experience in the lab and promises to remain with me for my future scientific ventures. I would also like to Dr. Daniel Liebler for imparting his mass spectrometry wisdom early in my graduate experience. His mentoring (and book, “Introduction to Proteomics”) allowed me to quickly grasp the basic principles and application of mass spectrometry to biological sciences. I also thank Dr. Michelle Dennehy, a post-doctoral fellow in his lab, who taught me the basics of instrument operation and trained me in affinity peptide enrichment strategies as well as Sheryl Stamer for facilitating the continued collaborator with the Liebler lab. I thank Dr. Harry Heijnen, a wonderful collaborator whose expertise in immunoelectron microscopy was pushed to the limit with SNO-protein detection…I thank him for his patience. I would like to thank both the Wistar Institute proteomics facility and CHOP protein core for accommodating my requests for every piece of raw data I generated; no matter how many hard drives it filled. Specifically, I would like to thank Tom Beer and Kaye Speicher at the Wistar Institute for their helpful discussions on proteomic analysis of large-scale datasets. I am thankful for the support of Lynn Spruce, Jessica Lee, and Hua Ding at the CHOP protein core, whose technical expertise in and patience for mass spectrometric instrumentation is unmatched. Importantly, I would like to thank the director of the protein core, Dr. Steven Seeholzer, who has taught me a great deal in the relatively iv short time we have been colleagues. Our computer programming sessions have benefited me tremendously by increasing the speed and efficiency with which I can analyze multiple sets of data. I would also like to thank Ethan Hughes, a fellow graduate student, whose enthuisiasm for astrocytes was so great I couldn’t help but incorporate them into my dissertation work. His knowledge of astrocyte biology provided fruitful discussions for designing future experiments. I would like to give specific recognition to Dr. Sarah Keene for teaching me all about astrocyte isolation and cell culture and for joining our expertise in preparation of the astrocyte secretome manuscript. And last, but certainly not least, I would like to thank all the other members of the Ischiropoulos lab that I have worked with throughout the years; Dr. Roberto Hodara, Dr. Ioannis Parastatidis, Dr. Joseph Mazzulli, Dr. Leonor Thomson, Dr. Paschalis-Thomas Doulias, Dr. Margarita Tenopoulou, Dick Lightfoot, Elpida Tsika, Marissa Martinez, Kristin Malkus, Jennifer Greene, Dr. Lindsay Johnston, and Dr. Christie Burno; all your support and friendship, regardless of whether experiments were a success or failure, has carried me through the years. v ABSTRACT MASS SPECTROMETRY-BASED PROTEOMICS REVEALS DISTINCT MECHANISMS OF ASTROCYTE PROTEIN SECRETION Todd Michael Greco Harry Ischiropoulos, Ph.D. The ability of astrocytes to secrete proteins subserves many of its known function, such as synapse formation during development and extracellular matrix remodeling after cellular injury. Protein secretion may also play an important, but less clear, role in the propagation of inflammatory responses and neurodegenerative disease pathogenesis. While potential astrocyte-secreted proteins may number in the thousands, known astrocyte-secreted proteins are less than 100. To address this fundamental deficiency, mass spectrometry-based proteomics and bioinformatic tools were utilized for global discovery, comparison, and quantification of astrocyte-secreted proteins. A primary mouse astrocyte cell culture model was used to generate a collection of astrocyte- secreted proteins termed the astrocyte secretome. A multidimensional protein and peptide separation approach paired with mass spectrometric analysis interrogated the astrocyte secretome under control and cytokine-exposed conditions, identifying cytokine-induced secreted proteins, while extending the depth of known astrocyte- secreted proteins to 169. Several of these proteins were likely secreted by non- vi conventional mechanisms. These non-conventional mechanisms were explored further using stable isotope labeling by amino acids in cell culture, revealing 12 putative non- conventionally secreted proteins. These qualitative and quantitative mass spectrometry approaches are broadly applicable for the study of cellular secretomes as well as for extension to in vivo secretomes. vii TABLE OF CONTENTS DEDICATION ............................................................................................. iii ACKNOWLEDGEMENTS ......................................................................... iv ABSTRACT ................................................................................................... vi TABLE OF CONTENTS .......................................................................... viii LIST OF TABLES ........................................................................................ xi LIST OF FIGURES .................................................................................... xii ABBREVIATIONS ...................................................................................... xv CHAPTER 1: BACKGROUND 1.1 Introduction ................................................................................... 1 1.2 Cellular pathways of protein secretion ....................................... 3 1.3 Astrocyte secretion of biomolecules ............................................. 7 1.4 Analysis of complex biological protein mixtures by mass spectrometry ................................................................................
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