FACTORS AFFECTING THE FRAGMENTATION OF PEPTIDE IONS: METAL CATIONIZATION AND FRAGMENTATION TIMESCALE A Dissertation by KEVIN LEE KMIEC 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 August 2012 Major Subject: Chemistry Factors Affecting the Fragmentation of Peptide Ions: Metal Cationization and Fragmentation Timescale Copyright 2011 Kevin Lee Kmiec FACTORS AFFECTING THE FRAGMENTATION OF PEPTIDE IONS: METAL CATIONIZATION AND FRAGMENTATION TIMESCALE A Dissertation by KEVIN LEE KMIEC 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 E. Hardin Simon W. North Emile A. Schweikert Head of Department, David H. Russell August 2012 Major Subject: Chemistry iii ABSTRACT Factors Affecting the Fragmentation of Peptide Ions: Metal Cationization and Fragmentation Timescale. (August 2012) Kevin Lee Kmiec, B.A., Hendrix College Chair of Advisory Committee: Dr. David H. Russell The factors affecting peptide fragmentation have been extensively studied in the literature in order to better predict the fragment ion spectra of peptides and proteins. While there are countless influences to consider, metal cation binding in the gas-phase is particularly interesting. Herein, a comparison of fragmentation patterns of a model peptide series with various charge carriers (H+, Li+, Na+, K+, and Cu+) will assist in determining the location of the preferred binding site of the metal cation and in assessing differences in the fragmentation pattern as a result of this binding site. An interesting observation from these studies reveals abundant x-type fragment ions occurring from the fragmentation of alkali-metal cationized peptides. As these fragment ions have been observed in previous studies by others but not addressed, the factors affecting the formation of these x-type fragment ions are explored. Additionally, a home-built 193-nm photodissociation tandem time-of-flight mass spectrometer is utilized to study how peptide fragmentation kinetics affect the fragmentation pattern observed. Initially, the fragmentation timescales of various peptides are investigated. Results indicate that longer fragmentation timescales (~10 iv microseconds) result in an increased number of identified peaks with internal and ammonia loss fragment ions being the most common in comparison to ‘prompt’ fragmentation timescales (~1 microsecond). Furthermore, b-type fragment ion formation is also favored at longer timescales for the arginine containing peptides investigated. The fragmentation pattern of several proline containing peptides is examined by collision-induced dissociation and 193-nm photodissociation. Unique fragment ions are observed with each occurring at a proline residue. Few differences are detected between CID and 193-nm photodissociation spectra, indicating that the proline residues direct fragmentation rather than the dissociation method. In an effort to improve the performance of the photodissociation tandem TOF instrument, the addition of a second source and a dual-stage reflectron are incorporated. The modifications result in improved mass range, signal-to-noise, and increased fragment ion collection efficiencies. High quality mass spectra are acquired across a range of mass-to-charge ratios from ~600 to 1900. Furthermore, the modifications continue to allow investigation of various fragmentation timescales with the addition of an additional timeframe of ~3 microseconds. v DEDICATION This dissertation is dedicated first and foremost to my family, my parents Edward and Janis, and my brother Kyle. The values my parents have taught me throughout my life have made me into the person I am today. My mom has always listened whenever life has gotten me down, and without her advice those down times would have been much more numerous and extensive. My dad, more than anyone else, has taught me the value of a hard day’s work and has always been there when I need a distraction from the sometimes tireless work of graduate school. Kyle is not just my brother; he is also one of my best friends and is still one of the few people in the world that can always make me laugh. This work is also dedicated to the love of my life and my future wife Cassie. She is always there to encourage me even when life is at its worst and without her consistent support I would never have reached this point in my life. She truly makes every day brighter. vi ACKNOWLEDGEMENTS I would like to first thank my advisor Dr. David H. Russell for providing me with the opportunity to earn a Ph.D. degree. This has been both a financial contribution and a physical contribution through his assistance within the laboratory. As an instrument builder, the progress at times can be slow, but with Dr. Russell encouragement, I persevered through most of the trials and tribulations without much delay. I am also thankful for the guidance and support of my committee members, Dr. Emile Schweikert, Dr. Simon North and Dr. Paul Hardin. I am especially thankful to the machinists, Will Seward and Carl Johnson, who have helped to create the instrumentation utilized in these studies. Without their skill set, my instrument is just a simple block of metal with to purpose or function. Their work is further enhanced by that of Greg Matthijetz. Without Greg’s consistent aid with electronics and Labview programming of the photodissociation time-of-flight instrument experiments would be far more difficult than could be imagined. My first years in graduate school would not have been possible without the excellent guidance of Dr. Garrett Slaton and Dr. Jody May. Garrett was a tremendous help during my first summer of work in the Russell group as he showed me the ropes of one of the more difficult experiments in the Russell Group, photodissociation tandem time-of-flight mass spectrometry. Jody inspired me to become more interested in mass spectrometry instrument development and modification which through the years has led vii me to construct an instrument far different from the version with which I originally began. Finally, I would like to thank the other former and current members of the Russell research group who have helped me through graduate school both in and out of the lab, specifically, Dr. Kent Gillig, Dr. Bill Russell, Dr. Shane Tichy, Dr. Brad Williams, Dr. Ryan Blase, Dr. Stephanie Cologna, Roberto Gamez, Kyle Fort, Josh Silveira, Junho Jeon, Fred Zinnel, Kim May, and Dr. Chaminda Gamage. I would also like to thank Dr. Francisco A. Fernandez-Lima who performed all theoretical calculations included in this work which yields a perfect complement to the fragmentation data shown herein. viii NOMENCLATURE ADC Analog-to-Digital Converter CID Collision-induced Dissociation COM Center-of-mass DC Direct Current ECD/ETD Electron Capture Dissociation/ Electron Transfer Dissociation ESI Electrospray Ionization FTICR Fourier Transform Ion Cyclotron Resonance IVR Intramolecular Vibrational Redistribution KE kinetic energy LC Liquid Chromatography LDI Laser Desorption Ionization m/z mass-to-charge ratio MALDI Matrix Assisted Laser Desorption/ Ionization MS Mass Spectrometry MCP Microchannel Plate PSD post source decay PTMs post translational modifications QET Quasi-equilibrium theory RF Radio Frequency RRK Rice, Ramsperger, Kassel ix RRKM Rice, Ramsperger, Kassel, Marcus S/N signal to noise SID Surface Induced Dissociation TDC Time-to-Digital Converter TIS timed ion selector TOF time-of-flight UV Ultraviolet x TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi NOMENCLATURE .................................................................................................. viii TABLE OF CONTENTS .......................................................................................... x LIST OF FIGURES ................................................................................................... xiii LIST OF TABLES .................................................................................................... xx CHAPTER I INTRODUCTION ................................................................................ 1 Ionization Methods ......................................................................... 2 Mass Analyzers .............................................................................. 6 Detectors and Signal Analysis Techniques .................................... 20 Gas-Phase Ion Fragmentation ........................................................ 24 Biological Mass Spectrometry: Proteomics ................................... 26 Factors Affecting Peptide Ion Fragmentation ................................ 29 II HOW DO METAL ION AFFINITY AND BINDING SITE INFLUENCE THE FRAGMENTATION OF PEPTIDE IONS?. ....... 47 Introduction .................................................................................... 48 Methods .......................................................................................... 51 Results and Discussion................................................................... 53 Conclusions ...................................................................................