ISOELECTRIC TRAPPING AND MASS SPECTROMETRY: TOOLS FOR PROTEOMICS A Dissertation by STEPHANIE MARIE COLOGNA Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2010 Major Subject: Chemistry ISOELECTRIC TRAPPING AND MASS SPECTROMETRY: TOOLS FOR PROTEOMICS A Dissertation by STEPHANIE MARIE COLOGNA Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, David H. Russell Committee Members, Paul S. Cremer Arul Jayaraman Gyula Vigh Head of Department, David H. Russell December 2010 Major Subject: Chemistry iii ABSTRACT Isoelectric Trapping and Mass Spectrometry: Tools for Proteomics. (December 2010) Stephanie Marie Cologna, B.S., University of Arizona Chair of Advisory Committee: Dr. David H. Russell Mass spectrometry (MS) has played a major role in the proteomic analysis of an array of biological samples. Even so, inherent limitations exist such as sample complexity and the dynamic range. In an attempt to overcome these limitations, pre- fractionation is typically performed followed by reversed phase liquid chromatography coupled with MS. Pre-fractionation can be performed in several formats including chromatographic or electrophoretic based methods. Solution-based isoelectric point (pI) fractionation, specifically isoelectric trapping (IET), provides an attractive alternative for pre-fractionation in bottom-up proteomic studies. A recently developed device, membrane separated wells for isoelectric focusing and trapping (MSWIFT), provides rapid separation on the basis of pI and resulting solutions are MS compatible without the need for extensive sample cleanup. Initial experiments demonstrate fractionation using MSWIFT, of peptide mixtures ranging from standards to a yeast lysate where resulting fractions are analyzed using matrix- assisted laser desorption/ionization (MALDI) – MS or further separated using reversed phase liquid chromatography followed by tandem MS (MS/MS) analysis. Identified yeast proteins range in size, pI and copy number illustrating an ability to increase the iv depth of proteome coverage when using MSWIFT. Extensive studies were also performed using MSWIFT in a multi-stage fractionation platform to improve peptide and protein identifications for the first large-scale proteomic study of the model fungus, Neurospora crassa. A second focus of this work is the development of a new sample preparation method for proteolytic digestion and high-throughput separations using MSWIFT. Histidine is used as a neutral pH, isoelectric, sample buffer for tryptic digestion of proteins and also assists in rapid separations using MSWIFT owing to the low conductivity. Tryptic digests of individual standard proteins and a mixture of standard proteins are used to illustrate these advantages. Finally, the histidine buffer sample preparation method is incorporated into a two-dimensional separation strategy. Tryptic peptides are fractionated using MSWIFT and resulting solutions are further separated using capillary electrophoresis (CE) coupled with MALDI-MS/MS. Performing the two-dimensional strategy allows for increased confidence in peptide and protein assignment owing to experimentally determined in-solution charge states and estimated pI values. v ACKNOWLEDGEMENTS I would first like to acknowledge and thank my advisor, Dr. David H. Russell for his continual support and guidance and for providing me the opportunity to perform research in his laboratory. He encouraged my individual research, welcomed ideas and spent many hours helping me develop as a scientist. For these things and many others, I am greatly indebted. I am also very appreciative of the guidance that was provided by Dr. Gyula Vigh and his willingness to open his laboratory to me. He is a great teacher and mentor and his excitement for chemistry is contagious. I have learned from him it is important to take a step back and think about the big picture and I am grateful for the many scientific and life lessons he has provided. Sincere thanks to the members of the Russell Research Group both past and present, for their help and guidance. I would especially like to thank Dr. William K. Russell for being an outstanding mentor and for providing me with endless opportunities to expand my knowledge. I would also like to thank Dr. Stacy D. Sherrod for her friendship, constant support, encouragement and willingness to help develop ideas and critically evaluate my work. A special thank you to Dr. Jody C. May for being a great friend and providing a hand when I needed to construct a piece or fix an electrode and for his willingness to give me a lesson on an array of topics. I would like to acknowledge my friend and coworker, Dr. Brad J. Williams for his support, everlasting patience and for teaching me many valuable things along the way. vi Finally, I would like to thank my undergraduate research advisor, Dr. Katrina M. Miranda for encouraging me to pursue my interests in chemistry, and to savor the joys of scientific research. vii NOMENCLATURE MS Mass Spectrometry MS/MS Tandem Mass Spectrometry MSWIFT Membrane Separated Wells for Isoelectric Focusing and Trapping IET Isoelectric Trapping IEF Isoelectric Focusing pI Isoelectric Point LC Liquid Chromatography MALDI Matrix Assisted Laser Desorption/Ionization ESI Electrospray Ionization CE Capillary Electrophoresis SCX Strong Cation Exchange HILIC Hydrophilic Interaction Liquid Chromatography GELLFrEE Gel-eluted Liquid Fraction Entrapment Electrophoresis GeLC Gel Electrophoresis – Liquid Chromatography MW Molecular Weight SDS Sodium Dodecylsulfate PAGE Polyacrylamide Gel Electrophoresis CIEF Capillary Isoelectric Focusing 2D-GE Two-Dimensional Gel Electrophoresis MCE Multicompartment Electrolyzer viii IPG Immobilized pH Gradient CID Collision Induced Dissociation ix TABLE OF CONTENTS Page ABSTRACT.............................................................................................................. iii ACKNOWLEDGEMENTS ...................................................................................... v NOMENCLATURE.................................................................................................. vii TABLE OF CONTENTS.......................................................................................... ix LIST OF FIGURES................................................................................................... xi LIST OF TABLES .................................................................................................... xvii LIST OF SCHEMES................................................................................................. xviii CHAPTER I INTRODUCTION................................................................................ 1 Mass Spectrometry-based Proteomics ........................................... 1 Pre-fractionation for Proteomic Studies......................................... 3 Multi-dimensional Protein Identification Technology................... 4 Electrophoretic Separations............................................................ 5 Isoelectric Focusing (IEF).............................................................. 6 Isoelectric Trapping (IET).............................................................. 10 Membrane Separated Wells for Isoelectric Focusing and Trapping (MSWIFT)...................................................................................... 14 II COMBINING ISOELECTRIC POINT-BASED FRACTIONATION, LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY TO IMPROVE PEPTIDE DETECTION AND PROTEIN IDENTIFICATION ............................................................................ 17 Introduction .................................................................................... 17 Experimental Section ..................................................................... 20 Results and Discussion................................................................... 26 Conclusions .................................................................................... 44 x CHAPTER Page III MUTLI-STAGE, ISOELECTRIC POINT-BASED SEPARATIONS FOR THE PROTEOMIC ANALYSIS OF THE MODEL FUNGUS: NEUROSPORA CRASSA ..................................................................... 46 Introduction.................................................................................... 46 Experimental Section ..................................................................... 48 Results and Discussion................................................................... 53 Conclusions .................................................................................... 61 IV STUDIES OF HISTIDINE AS AN ALTERNATIVE BUFFER FOR TRYPTIC DIGESTION AND ISOELECTRIC TRAPPING FRACTIONATION IN BOTTOM-UP PROTEOMICS ..................... 62 Introduction .................................................................................... 62 Experimental Section ..................................................................... 64 Results and Discussion................................................................... 66 Conclusions .................................................................................... 77 V USING HISTIDINE BUFFER FOR PROTEOMICS: COUPLING MSWIFT FRACTIONATION AND CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY FOR HIGH THROUGHPUT PROTEOMIC ANALYSES .................................. 78 Introduction ...................................................................................