Comparison of Label and Label-free Quantitative Liquid Chromatography Tandem Mass Spectrometry for Protein Biomarker Discovery THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Bei Zhao Graduate Program in Chemistry The Ohio State University 2010 Master's Examination Committee: Dr. Michael A. Freitas, Advisor Dr. Susan V. Olesik, Co-advisor Copyright by Bei Zhao 2010 ABSTRACT Mass spectrometry-based protein quantification approaches are powerful tools for biomarker discovery. In this thesis, a spectral counting-based label-free analysis platform and tandem mass tag isobaric labeling quantification platform are described and evaluated for detecting differential protein abundances of an in vivo system in response to UV radiation. Sample preparation and separation of global protein digests were optimized for both label-free and labeling methods. Statistical evaluations such as principal component analysis and Venn diagram were used to evaluate the reproducibility of the analytical system. For spectral counting analysis, a few different normalization and modification methods such as ratioing of normalized spectral counting, normalized spectral abundance factors, and spectral index were applied and the results were compared. Correlations of the results from spectral counting and tandem mass tags labeling were also carried out. Potential protein biomarkers related to DNA damage repair in the in vivo system are proposed based on both labeling and label-free approaches. ii DEDICATION This document is dedicated to my parents iii ACKNOWLEDGMENTS I wish to express sincere thanks to my advisors Dr. Michael A. Freitas and Dr. Susan Olesik for their valuable guidance and support. I also want to thank all of the group members and collaborators in this research. Especially I would like to thank Dr. Liwen Wang for training me in the lab, Dr. George Heine and Dr. Jeffrey Parvin, the collaborators for the project in the thesis, Nan Kleinholz from Campus Chemical Instrument Center who offered a lot of help on instrumentation. I also would like to thank Mr. John Shapiro, Jonathan Clark and Josh Dettman for discussion. Special thanks to my dearest parents and my best friend Renan Cabrera, who supported me mentally and financially without any reservations. The study was funded by the Ohio State University. iv VITA June 2003 .......................................................B.S. Marine Chemistry, Ocean University of China, China March 2006 ...................................................M.S. Physical Chemistry, University of Windsor, Canada 2006 to present ..............................................Graduate Teaching and Research Assistant, Analytical Chemistry, Department of Chemistry, The Ohio State University PUBLICATIONS 1. Bei Zhao, Jichang Wang. “Chemical Oscillations during the Photoreduction of 1, 4- benzoquinone in Acidic Bromate Solution” J. Photochem. Photobiol. A: Chem 2007, 192, 204-210. 2. Bei Zhao, Jichang Wang. “Photomediated bromate-1, 4-benzoquinone reaction: A novel photochemical oscillator” Chem. Phys. Lett. 2006, 430, 1-3, 41-44. 3. Bei Zhao, Jichang Wang.“Stirring-Controlled Bifurcations in the 1, 4-Cyclohexanedione- Bromate Reaction” J. Phys. Chem. A. 2005, 109, 16, 3647-3651. 4. Jichang Wang, Krishan Yadav, Bei Zhao, QingYu Gao, Do Sung Huh. “Photo- Controlled Oscillatory Dynamics in the Bromate-1, 4-Cyclohexanedione Reaction” J. Chem. Phys. 2004, 121, 10138-10144. v FIELDS OF STUDY Major Field: Chemistry vi TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii DEDICATION ................................................................................................................... iii ACKNOWLEDGMENTS ................................................................................................. iv VITA ................................................................................................................................... v TABLE OF CONTENTS .................................................................................................. vii LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ........................................................................................................... xi CHAPTER 1 INTRODUCTION ........................................................................................ 1 1.1 Overview of quantification by mass spectrometry .................................................... 1 1.2 Isotope-labeled mass spectrometry ........................................................................... 2 1.2.1 Isotope-labeled mass spectrometry ..................................................................... 2 1.2.2 Advantages and limitations of isotope-labeled mass spectrometry .................... 2 1. 3 Label-free mass spectrometry approaches ............................................................... 3 1.3.1 Label-free mass spectrometry approaches .......................................................... 3 vii 1.3.2 Peptide chromatographic peak intensity measurements ..................................... 4 1.3.3 Spectral counting quantification ......................................................................... 4 1.3.4 Advantages and limitations of label-free approaches ......................................... 6 1.4 Summary ................................................................................................................... 7 CHAPTER 2 DEVELOPMENT OF ROBUST LABEL-FREE PROTEOMICS TO DETERMINE PROTEIN CHANGES IN UV-INDUCED DNA DAMAGE .................... 8 2.1 Introduction ............................................................................................................... 8 2.1.1 Spectral counting label-free quantification approach ......................................... 9 2.1.2 The TMT quantification approach .................................................................... 11 2.2 Experimental ........................................................................................................... 12 2.2.1 Material ............................................................................................................. 12 2.2.2 Cell culture and treatments ............................................................................... 13 2.2.3 Sample preparation for label-free analysis ....................................................... 13 2.2.4 Sample preparation of TMT isotope-labeled peptides ..................................... 17 2.2.5 LC-MS/MS analysis ......................................................................................... 22 2.2.6 Database search ................................................................................................ 23 2.3 Results and discussion ............................................................................................. 25 2.3.1 Separation gradient optimization ...................................................................... 25 2.3.2 Evaluation of the reproducibility of the chromatography ................................ 27 viii 2.3.3 Comparison analysis of the number of identified proteins ............................... 42 2.3.4 Data Reduction ................................................................................................. 60 2.3.5 Normalization of label-free spectral count data ............................................... 77 2.3.6 Analysis with spectral counting method ........................................................... 78 2.3.7 Analysis by use of Spectral Index (SI) ............................................................. 81 2.3.8 Analysis by use of normalized spectral abundance factors .............................. 85 2.3.9 Normalization for TMT data ............................................................................ 90 2.3.10 Analysis of the TMT data ............................................................................... 91 2.3.11 Correlation of label-free spectral counting data and TMT data ..................... 93 2.3.12 Cluster analysis ............................................................................................... 95 2.3.13 Potential biomarker selection ....................................................................... 100 2.4 Conclusion ............................................................................................................. 109 CHAPTER 3 SUMMARY .............................................................................................. 111 REFERENCES ............................................................................................................... 112 APPENDICES ................................................................................................................ 119 Appendix A: ZipTip clean-up procedure ................................................................. 119 Appendix B: Tables ................................................................................................. 120 ix LIST OF TABLES Table 2.1 Potential protein biomarkers for HeLa cell samples. ...................................... 105 Table 2.2 Potential protein biomarkers for MCF-10A cell samples. .............................. 107 Table B.1 Ratios and p-values for the t-test of log2(NSAF) for selected HeLa cell protein biomarker………………………………………………………...………………….….119