DEVELOPMENT OF METHODS FOR MASS SPECTROMETRY ANALYSIS OF PROTEINS IN SUPERNATANTS HARVESTED FROM IN VITRO HUMAN LUNG CELLS (A549) by Edward Gee-Ming Lau B.Sc., Simon Fraser University, 2006 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Chemistry © Edward G. Lau 2010 SIMON FRASER UNIVERSITY Fall 2010 All rights reserved. However, in accordance with the Copyright Act of Canada, this work may be reproduced, without authorization, under the conditions for Fair Dealing. Therefore, limited reproduction of this work for the purposes of private study, research, criticism, review and news reporting is likely to be in accordance with the law, particularly if cited appropriately. APPROVAL Name: Edward Gee-Ming Lau Degree: Master of Science Title of Thesis: Development of Methods for Mass Spectrometry Analysis of Proteins in Supernatants Harvested from in vitro Human Lung Cells (A549) Examining Committee: Chair: Dr. Robert Britton Assistant Professor ______________________________________ Dr. George Agnes Senior Supervisor Associate Dean of Graduate Studies Professor – Department of Chemistry ______________________________________ Dr. Paul Li Supervisor Professor – Department of Chemistry ______________________________________ Dr. Charles Walsby Supervisor Associate Professor – Department of Chemistry ______________________________________ Dr. Luis Sojo External Examiner Adjunct Professor – Department of Chemistry Date Defended/Approved: December 9, 2010 ii Declaration of Partial Copyright Licence The author, whose copyright is declared on the title page of this work, has granted to Simon Fraser University the right to lend this thesis, project or extended essay to users of the Simon Fraser University Library, and to make partial or single copies only for such users or in response to a request from the library of any other university, or other educational institution, on its own behalf or for one of its users. The author has further granted permission to Simon Fraser University to keep or make a digital copy for use in its circulating collection (currently available to the public at the “Institutional Repository” link of the SFU Library website <www.lib.sfu.ca> at: <http://ir.lib.sfu.ca/handle/1892/112>) and, without changing the content, to translate the thesis/project or extended essays, if technically possible, to any medium or format for the purpose of preservation of the digital work. The author has further agreed that permission for multiple copying of this work for scholarly purposes may be granted by either the author or the Dean of Graduate Studies. It is understood that copying or publication of this work for financial gain shall not be allowed without the author’s written permission. Permission for public performance, or limited permission for private scholarly use, of any multimedia materials forming part of this work, may have been granted by the author. This information may be found on the separately catalogued multimedia material and in the signed Partial Copyright Licence. While licensing SFU to permit the above uses, the author retains copyright in the thesis, project or extended essays, including the right to change the work for subsequent purposes, including editing and publishing the work in whole or in part, and licensing other parties, as the author may desire. The original Partial Copyright Licence attesting to these terms, and signed by this author, may be found in the original bound copy of this work, retained in the Simon Fraser University Archive. Simon Fraser University Library Burnaby, BC, Canada Last revision: Spring 09 ABSTRACT Proteins are primarily responsible for functionalizing cells and, with respect to an initiative in soft ionization mass spectrometry (MS), the relative abundances of selected proteins have been used as indicators of normal or stressed physiological states. Methods developed for this thesis describe the use of matrix assisted laser desorption ionization time of flight (MALDI-ToF) MS to monitor the ion signals of C-X-C motif chemokine 5 and ubiquitin protein. Identification of these ion signals were carried out by performing additional experiments using liquid chromatography interfaced to an electrospray ionization (LC-ESI) equipped MS capable of tandem MS. A total of 78 proteins were identified by LC-ESI tandem MS, but not all corresponded to MALDI-MS data. In addition to the use of commercially available instrumentation, a separate study was performed to investigate the potential for an AC trap to generate molecular ions from a single levitated droplet having undergone Coulomb explosion. Keywords: Mass Spectrometry; Matrix Assisted Laser Desorption Ionization Time of Flight; Liquid Chromatography Electrospray Ionization; Tandem MS; Protein Identification; Single Droplet Levitation iii DEDICATION For my Mommy and Grandma iv ACKNOWLEDGEMENTS I am grateful beyond adequate acknowledgement to my supervisor George Agnes for his mentorship and guidance from my latter undergraduate semesters through my graduate studies. He provided me with a chance to advance in chemistry by showing me areas of research previously unknown to me. I remember him telling me just before I started my MSc., ―You remind me of myself back when I was in school, just doing alright in school until you find an area you want to excel in.‖ I consider myself extremely fortunate to have been a student under his supervision, conducting research in an environment that allowed me to learn and investigate ideas with a tremendous amount of freedom. I would like to thank my fellow lab members over the years who have helped me learn and provide insightful comments related to my research. Particularly, I thank Allen Haddrell (teaching me how to operate a MALDI-ToF and culture cells), Xinzhou (Siry) Hu (expanding my cell culturing abilities), Neil Draper (constant helpful insight), Mariana (helpful insight in to molecular biochemistry explained to a chemist), and of course the many others who helped make the Agnes Lab a comfortable second home. I am thankful for the support of the British Columbia Proteomic Network. Through this organization I could access a licensed version of Mascot software, and make use of Thomas Clark‘s (MS service specialist) expertise. Thanks to v Tom, he taught me how to assemble in-house an online nano-flow LC-ESI ion source, and prepared capillary reverse phase columns for me to use. Lastly I have to thank my family, particularly my mom and grandmother for their strong belief in education, and their sacrifices for me to obtain one. I respect them more than anyone for their courage, conviction, and hard work for immigrating from their home in China, to Hong Kong, and ultimately Canada. I also thank my big sister and the rest of my family for a combination of their tolerance and enlightenment. Thank you for tolerating and entertaining what I can now imagine were endless annoying queries of ―Why‘s and How‘s‖, at the same time enlightening me with ―Did you know‘s and Want to see‖. vi TABLE OF CONTENTS Approval .......................................................................................................................... ii Abstract .......................................................................................................................... iii Dedication ...................................................................................................................... iv Acknowledgements ......................................................................................................... v Table of Contents .......................................................................................................... vii List of Figures.................................................................................................................. x List of Tables .................................................................................................................xiv Glossary ........................................................................................................................ xv 1: A Brief Perspective of Proteomics, Human Inhalation of Ambient Particulate Matter, and Analytical Determinations of Proteins .................................. 1 1.1 Proteomics and its Applications ............................................................................... 1 1.2 Particulate Matter and Human Health ...................................................................... 2 1.2.1 Particulate Matter and Human Epidemiology ............................................... 2 1.2.2 Particulate Matter ........................................................................................ 3 1.2.3 Inhalation of Particulate Matter into the Lung ............................................... 4 1.3 Established Techniques for Protein Analysis ........................................................... 5 1.3.1 Properties of Proteins for Separation ........................................................... 6 1.3.2 Methodologies for Detecting Proteins .......................................................... 9 1.3.3 Edman Degradation for Sequencing Amino Acids ..................................... 11 1.4 Characterization of Proteins by Mass spectrometry ............................................... 12 1.4.1 Protein Identification by MS ....................................................................... 12 1.4.2 Quantitative Proteomics ............................................................................