1 Development of Sensitive High Performance Analytical Methods for the Comprehensive Characterization of Proteins and Glycoproteins from Samples of Clinical and Biopharmaceutical Importance A dissertation presented by Dipak A. Thakur to The department of Chemistry and Chemical Biology In partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of Chemistry Northeastern University Boston, Massachusetts June 2011 2 Development of Sensitive High Performance Analytical Methods for the Comprehensive Characterization of Proteins and Glycoproteins from Samples of Clinical and Biopharmaceutical Importance by Dipak A. Thakur ABSTRACT OF DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate School of Arts and Sciences of Northeastern University, June 2011 3 ABSTRACT This thesis focuses on the development of ultra sensitive high resolution analytical methods for the characterization of proteins and glycoproteins from samples of clinical and biopharmaceutical origin. In the first instance the combination of laser capture micro dissection (LCM) for the selective enrichment of homogenous but low number cell populations in combination with down-stream porous layer open tubular column (PLOT) liquid chromatography-mass spectrometry (LC-MS) using both one- and two-dimensional separations is described. The second portion of the thesis describes the ultra high performance analysis of intact recombinant a-human chorionic gonadotrophin glycoforms using capillary electrophoresis with accurate mass high resolution Fourier transform ion cyclotron resonance mass spectrometry (CE-FTMS). In Chapter 1 an overview of current analytical methods and technologies applied in the field of proteomics is discussed. A critique of these technologies is also performed laying down the foundations for the developments and improvements in current state-of- the-art as presented in the subsequent Chapters. In Chapter 2 the development of a micro-proteomic workflow for the comprehensive analysis of just 10,000 cells, collected by LCM, from invasive and metastatic epithelial cell types from a breast cancer patient is described. To minimize sample loss the development of an efficient sampling handling approach was necessary. To achieve this protein level separation and subsequent enzymatic digestion of the cell lysate was performed using short distance SDS-PAGE separation on tricine-PAGE gels. By combining this sample clean-up and fractionation approach with ultrasensitive 1D PLOT LC-MS in excess of 1,000 proteins were identified following injection of just 4 1/10th of the digested lysate or approximately 1,000 cells. The micro-proteomic workflow is highly suited for the comparative analysis of such small but highly informative LCM collected cell populations, more than 100 proteins were found to be differentially expressed thereby facilitating a deeper understanding of the associated biological changes associated with the invasive to metastatic transition. In Chapter 3 the application of an online 2D-RP/SCX/SPE/PLOT LC-FT-MS micro- proteomics platform is presented for the comparative proteomic analysis of LCM collected normal and triple negative breast cancer cell population. Using the effective sample handling approach described in Chapter 2 followed by fractionation and ultra sensitive analysis of the lysate, the tryptic digest corresponding to 4,000 cells using the 2D-RP/SCX/SPE PLOT LC-FT-MS platform in excess of 15,000 unique peptides corresponding to 4,259 proteins were identified. This deep proteome coverage further emphasizes the utility of the developed micro-proteomic platform for the analysis of trace quantities of proteins generated from small but highly biologically important LCM enriched cell populations. In chapter 4 the development and application of a high resolution CE-FTMS method for intact glycoform profiling of recombinant α-human chorionic gonadotrophin is described. The CE separation parameters used allowed for the rapid analysis, <20 minutes, and high resolution of >60 different glycoforms bearing up to nine sialic acids in addition to other glycoforms differing by the number and extent of uncharged monosaccharides. A low volume pressurized liquid junction, which preserves the high resolution of the CE separation, was used to interface the CE system with high resolution FTMS thereby allowing accurate determination of charge state and accurate mass of each intact 5 glycoform following deconvolution. In addition to the intact glycoform, profiling analysis of glycopeptides and glycans was also performed to determine and assign the population of oligosaccharides present at each individual glycosite, thereby facilitating complete and comprehensive characterization of r-ahCG. The methodology developed in Chapter 4 was further applied to the analysis of r-αhCG from different expression systems, CHO and murine cell based. The CE-FTMS method is readily applicable for characterization of drug substance/product as well as in process monitoring of these complex glycoforms. 6 ACKNOWLEDGEMENT I want to express my sincere and heartfelt gratitude to many people, teachers, colleagues and friends, who have helped me in reaching this milestone. First, I would like to acknowledge my thesis advisor, Professor Barry L. Karger, for accepting me as his student and giving me an opportunity to work in his research group. His guidance was constructive and aimed at bringing best out of me as a scientist and a person. Importantly, I was inspired and motivated by his wisdom, enthusiasm and commitment to highest standards. I would like to thank Dr. Tomas Rejtar for devoting his time and energy while guiding me on various projects. I would like to appreciate Dr. Marina Hincapie, Dr. Andras Guttman, Dr. Billy Wu, Dr. Shujia Dai, Dr. Sanwon Cha and Dr. Jonathan Bones for sharing their knowledge and expertise. I would like to thank my dissertation committee members, Prof. Paul Vouros, Prof. Graham Jones and Prof. Roger Giese for their time, suggestions and guidance. Many thanks to Dr. Buffie Clodfelder-Miller (Cellular and Molecular Neuropathology Core, University of Alabama), Elizabeth Richardson, Shemeica Binns, Sonika Dahiya and Dennis Sgroi (Massachusetts General Hospital) for providing precious LCM samples. I would like to thank our collaborators N.Washburn, C.J. Bosques, N.S.Gunay, Z.Shriver, and G.Venkataraman (Momenta Pharmaceuticals) for supporting glycoform profiling project and for their full contribution towards the glycan analysis. I would like to acknowledge the support and friendship of current and former researchers of Barnett Institute, Dr. E.Moskovets, Dr. Vickor Andreev, Dr. Quanzhou Luo, Dr. 7 Guihua Yue, Mr. Laxmi Manohar Akella, Dr. Claudia Donnet, Dr. Enrique Avarelo, Dr. Zoltan Sabo, Dr. Jim Glick, Somak Ray; previous and current graduate students lingyun Li, Ye Gu, Dongdong Wang, Majlinda Kulloli, Agnes Rafalko, Jonna Linholm-Ventola, Jack Liu, Chen Li, Peter Li, Chris Morgan, Vaneet Sharma, Rose Gathungu, Joshua Klaene and Fateme Tousi. I would like to express my gratitude to Jeffrey Kesilman, Felicia Hopkins, Richard Pumphrey, Andrew Bean, Jana Volf and Bill O,Neil for their support. I would like to acknowledge my wife, Vaishali, daughter Radhika, and son Hrishikesh for their love, support, sacrifice and compromise during 5 long years. Many many thanks to my parents, Sudha and Arjun Thakur, for their support, encouragement and care. I would like to thank my brother, Ganesh and his family, for supporting, guiding and encouraging me during my graduate studies. I would like to express my gratitude to my sister Jyoti and her family for their support and encouragement. 8 TABLE OF CONTENTS ABSTRACT………………………………………………………………………. 3 ACKNOWLEDGEMENT………………………………………………………… 6 TABLE OF CONTENTS………………………………………………………….. 8 LIST OF FIGURES.…………………………………………………………….…..14 LIST OF TABLES……………………………………………………………..……16 LIST OF ABBREVIATIONS AND CONVENTIONS…….……………………….16 Chapter 1: Overview of Technologies and Methodologies for Proteomics Analysis…………………………………………………………………………..…19 1.1 Introduction………………………………………….…….…………………….20 1.1.1 Proteomics: An Overview………………………………………………….….20 1.2 Shotgun Proteomics Methodologies…………………………………………..…23 1.2.1 Samples………………………………………………………………………...25 1.2.1.1 In Vitro Sample Source: Cell lines…………………………………….….....25 1.2.1.2 In Vivo Sample Sources…………………………………….……………..…26 1.2.2 Tissue Microdissection………………………………….……………………...28 1.2.2.1 Laser Capture Microdissection………………………….……………….…...30 1.2.2.2 Laser Microbeam Microdissection (LMM) ….......................................... 32 1.2.2.3 Comparison of LCM and LMM……………………………..……………….33 1.2.3 Sample Preparation……………………………………………..………..….…34 1.2.3.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ……….……...…..36 1.2.4 Separation Techniques…………………………………………………….…...38 1.2.4.1 High Pressure Liquid Chromatography…………………………………..... 38 1.2.5 Mass Spectrometry…………………………………………………..………..40 1.2.5.1 Ionization Methods……………………………………………………….. 40 1.2.5.2 Mass Analyzers………………………………………………………….. 42 1.2.5.3 Database Searching Tools for Proteomics……………………………….. 47 1.3 Microproteomics………………………………………………………….. 54 1.3.1 Alternative strategies for protein digestion………………………………….. 56 1.3.1.1 Solvents based approach………………………………………………….. 56 1.3.1.2 Cleavable surfactant……………………………………………………….. 57 9 1.3.1.3 Filter-Aided Sample Preparation (FASP) ……………………………….. 59 1.3.2 High Performance Liquid Chromatography for Microproteomics………….