Comparative Proteomic Profiling and Biomarker Discovery in Complex Biological Samples by Mass Spectrometry By Di Ma A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmaceutical Sciences) at the UNIVERSITY OF WISCONSIN-MADISON 2012 Date of final oral examination: 11/19/12 The dissertation is approved by the following members of the Final Oral Committee: Lingjun Li, Professor, Pharmaceutical Sciences Albee Messing, Professor, Comparative Biosciences Manish Patankar, Associate Professor, Obstetrics and Gynecology Joel Pedersen, Professor, Soil Science Richard Peterson, Professor, Pharmaceutical Sciences i Acknowledgement I would first like to thank my advisor Professor Lingjun Li for her guidance and support throughout my thesis research. Lingjun’s enthusiasm for science, interest to explore new technologies combined with her broad knowledge from years of experience made a profound impact on me and motivated me to be a much better researcher than what I was content to be. I thank Lingjun for the opportunity to work in her lab and for guidance into “all things” analytical chemistry. I thank my thesis committee that includes Professors Albee Messing, Manish Patankar, Joel Pedersen and Dick Peterson. I appreciate their support, advice and instruction during my thesis work. I have been fortunate to participate in several collaborations with groups on and off campus. I thank Professor Manish Patankar, Drs. Arvinder Kapur and Weifeng Cao for the work we did together for proteomic analysis of NK cells. I thank Professor Judd Aiken, Drs. Allen Herbst and Xin Wei for the work we did together to identify prion disease biomarker. I thank Dr. Xudong (Daniel) Shi and Chenxi Yang for the work we did together for the secretome research. I am thankful for the experiences that I had working with so many excellent scientists. I thank my coworkers in the Li lab for all of their support. In particular, I thank former member Dr. Xin Wei for his mentorship after I joined lab. Without his support I could not have adapted to the lab in the quick manner as I did to begin my independent research. In addition, I would especially like to thank Dr. Rob Sturm, Dr. Robby Cunningham, Dr. Weifeng Cao, Dr. Chenxi Jia, Claire Schmerberg and Chris Lietz for their technical support whenever I ran into ii problems. I also enjoyed working with Chenxi Yang in the past few months. The discussions with her added more insights and inspiration into my research. I thank the Analytical Instrument Center at School of Pharmacy, in particular, Dr. Cameron Scarlett for access to the Bruker amaZon ion trap mass spectrometer. I thank Linda Frei, Ken Niemeyer, Joni Mitchell, Jenny Hergenrother and Tom O'Connor for administrative assistance. Last but not least, I thank my father Wu Ma and my mother Wei Jin for their undying love and support. My parents’ dedication to hard work inspired me to fulfill my dreams to make a positive impact to society. I thank my awesome husband Kong Xiong for his infinite love and support especially during setbacks throughout graduate school when nothing seemed reassuring but his words of encouragement. I could not have made through graduate school without him. I dedicate this thesis to my family and to the reunion with my “Big Cheeks”. iii Table of Contents Page ______________________________________________________________________________ Acknowledgements i Table of Contents iii Abstract iv Chapter 1: Introduction: Background and Research Summary 1 Chapter 2: Mass Spectrometry-Based Proteomics and Peptidomics for 9 Systems Biology and Biomarker Discovery Chapter 3: Differential Expression of Proteins in Naïve and IL-2 Stimulated Primary 67 Human NK Cells Identified by Global Proteomics Chapter 4: Comparative Secretome Analysis of Vascular Smooth Muscle Cells in 104 Response to Smad3-Dpendent TGF-β Signaling Chapter 5: Searching for Reliable Pre-mortem Protein Biomarkers for Prion 130 Diseases – Progress and Challenges to Date Chapter 6: Identification and Validation of a Potential Protein Biomarker for 169 Prion Diseases by Mass Spectrometry-Based Quantitative Glycoproteomics Chapter 7: Conclusions and Future Directions 207 Appendix 1: Selected Protocols for Comparative Proteomic Profiling and Biomarker 217 Discovery in Complex Biological Samples by Mass Appendix 2: List of IL-2 Regulated Proteins in Human Primary NK Cells Identified 229 From All Three Donors (Chapter 3) Appendix 4: List of Publications and Presentations 248 iv Comparative Proteomic Profiling and Biomarker Discovery in Complex Biological Samples by Mass Spectrometry Di Ma Under the supervision of Professor Lingjun Li at the University of Wisconsin-Madison Abstract Advances in mass spectrometry (MS) technology have made MS-based proteomics a promising tool for protein profiling and biomarker discovery in various types of biological samples such as cell cultures, tissues, and biological fluids. MS-based proteomics has been widely applied to molecular and cellular biology to elucidate biological and pathophysiological processes. However, MS analyses of biological samples are often challenging due to the vast complexity and large dynamic range. Because disease identifying biomarkers are more likely to be low abundance proteins, it is imperative to remove the highly abundant proteins or apply enrichment techniques during sample preparation to allow detection and improve coverage of the low abundance proteins for MS analysis. In addition, the complexity of the digested biological samples can be reduced by applying multiple orthogonal separations prior to LC-MS/MS such as multidimensional protein identification technology (MudPIT). In this dissertation, the major objectives include the following: (1) method development of sample preparation and chromatographic separation for MS-based quantitative proteomics, and (2) their applications in large-scale protein characterization in complex biological samples for differential expression analysis and biomarker discovery will be discussed. First, a method of MudPIT combined with ESI-MS/MS was optimized and performed for global proteome profiling in naïve and v interleukin-2 (IL-2)-activated natural killer cells. In order to identify IL-2 regulated proteins that may lead to the discovery of new molecular pathways involved in IL-2 signaling, spectral counting, which is a label-free quantification strategy for comparative proteomic analysis was utilized due to the simplicity and sensitivity of this approach. A similar sample preparation and quantification strategy was also applied to the comparative secretome analysis in rat vascular smooth muscle cells (VSMCs) stimulated by transforming growth factor-β (TGF-β), which led to the identification of secreted proteins that may be associated with TGF-β signaling in VSMCs. Although multi-dimensional separation proved to be essential and effective to reduce sample complexity, it is sometimes insufficient for proteome profiling in biofluids such as plasma where only a few high abundance proteins comprise majority of the serum proteome. To improve the identifications of low abundance proteins in MS analysis, it is imperative to remove the highly abundant proteins or apply enrichment techniques prior to MS analysis. To tackle this problem, lectin affinity chromatography (LAC) was utilized in sample preparation of mouse plasma affected by prion disease to specifically enrich glycoproteins that may prove to be important biomarkers for prion diseases. The combination of LAC and MudPIT significantly reduced sample complexity and led to the discovery of a panel of potential biomarkers including the validation of serum amyloid P-component (SAP). Furthermore, PNGase F digestion analysis confirmed that the glycosylated form of SAP could be used as a potential diagnostic biomarker for prion diseases and that glycosylated SAP plays an important role in the progression of prion disease. Collectively, the work included in this thesis extends the capability of mass spectrometry as a powerful analytical tool for large-scale proteomic analysis in complex biological samples to identify disease biomarkers or biomolecules involved in critical cellular processes. 1 Chapter 1 Introduction: Background and Research Summary 2 1.1 Introduction The main objective of this dissertation focuses on the utilization of mass spectrometry (MS) for large-scale proteomic profiling in complex biological samples and application of this technology to discover biomarkers or protein factors that are associated with human diseases. For this purpose, the research included in this dissertation includes topics ranging from analytical method development for improved protein characterization and quantification, to biological validation of potential protein biomarkers identified by MS analysis. In this dissertation MS- based proteomics techniques were applied to vastly different collaborative projects where analyses were challenged by sample complexity. This chapter outlines the overall structure of this dissertation with a background overview of MS-based proteomics and general introductions to each research project. 1.2 Background Overview Advances in MS have made MS-based proteomics and peptidomics a method of choice for large scale protein characterization and biomarker discovery. Coupled with different sample preparation and separation techniques, MS-based proteomics and peptidomics can be utilized to study peptide and protein identification, structures, modifications and interactions