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Discovery and Quantitation of Protein Modifications using Targeted Mass Spectrometry Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jia You, M.E. Graduate Program in Chemistry The Ohio State University 2012 Dissertation Committee: Dr. Michael A. Freitas, Advisor Dr. Dehua Pei, Co-Advisor Dr. Anne Co Copyright by Jia You 2012 Abstract In this dissertation, efforts were focused on the development of targeted proteomic assays to elucidate differences in protein profiles present in diseases and their correlation with other molecular markers (proteins or microRNA). In Chapter 2, a high-sensitivity TFA-free LC-MS method is described. The analysis of proteins by reversed-phase liquid chromatography (RPLC) commonly involves the use of TFA as an ion-pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC-MS, TFA-free methods for protein separation optimized. Protein standards and histones were used to evaluate TFA-free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C8 columns with the ion-pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients. ii In chapter 3, a targeted mass spectrometry approach was used to examine nitration and nitrosylation of tyrosine residues on tropomyosin. A highly versatile target-driven MS/MS strategy was developed to facilitate identification and quantification of especially low abundance protein post-translational modifications. The LC-MS/MS analysis was carried out on a LTQ-Orbitrap mass spectrometer to take advantage of its high mass resolution and high mass accuracy. A recursive process was used to discover, verify and quantify all the possible nitrated and nitrosylated peptides. Measurement of nitrotyrosine and nitrosyltyrosine on Tm highlights the utility of this approach for discovering and characterizing the challenging low abundant post-translational modifications. In Chapter 4, phosphotyrosine (pTyr) protein enrichment was used to assist the identification of new potentially druggable targets in FLT3 internal tandem duplication (FLT3 ITD) driven acute myeloid leukemia (AML). The MV4-11 cell line was used in this study because it carries the FLT3 ITD activating mutation. pTyr protein enrichment was used to characterize protein extracts from MV4-11 cells before and after treated with the tyrosine kinases inhibitor PKC412. The use of 4G10 anti-pTyr conjugated beads yielded high quality pTyr enrichment with low background. New FLT3 downstream signaling effectors were expected to be identified from pTyr immunopurified protein complex by LC-MS3 analysis, and serve as novel therapeutic targets in AML. iii In Chapter 5, DICER-associated or DGCR8-associated proteins were studied by LC-MS/MS following immunoprecipitation with Flag-DICER or Flag-DGCR8. Among the identified proteins, nucleolin (NCL), a major nucleolar protein often up-regulated in cancer, was detected and confirmed as a component of the DROSHA-DGCR8 complex by coimmunoprecipitation experiments, as previously reported. Further research was focused on this RNA-binding protein NCL to characterize its role in miRNA biogenesis. Experimental data showed NCL regulates the biogenesis of a specific cohort of miRNAs. Since miRNA level modulation as a therapeutic approach has been considered challenging so far, these findings could have a strong clinical impact on the development of future miRNA-based anti-cancer therapies. iv Dedication This document is dedicated to my mother, Wanhong Cao. v Acknowledgments I would like to thank my advisor, Dr. Michael. A. Freitas, for his instruction and support. His knowledge, experience and expertise helped me through my Ph.D study. Also, I appreciate him for providing me the opportunity to learn, think and work independently. The experience and training I obtained in Dr. Freitas group will benefit me greatly for the rest of my life. I would also like to thank Dr. Dehua Pei, my co-advisor in the Department of Chemistry. His support allowed me to pursue the research in which I felt most interested. I want to thank the group members including Dr. Hua Xu, Dr. Liwen Wang, Dr. Lanhao Yang, Dr. Kelly Telu, Xiaoyan Guan, Sean Harshman, Linan Wang, and Owen Branson for their support. I also want to thank my collaborators: Dr. John Byrd, Dr. Brandon Biesiadecki, Dr. Guido Marcucci, and Dr. Flavia Pichiorri. Their support and help were valuable to me. Thanks are extended to their group members. Many thanks to Dr. Kari Green-Church, Dr. Liwen Zhang and Nan Kleinholz for sharing instruments. vi I also would like to thank all the professors who taught me in graduate school. Special thanks to Dr. Susan. V. Olesik for the helpful discussion and comments on my chapter 2. vii Vita July 2002 ............................................ B.E. Biochemical Engineering, Nanjing University of Science & Technology, Nanjing, China December 2005 .................................. M.E. Biochemical Engineering, Nanjing University of Science & Technology, Nanjing, China September 2006 to present ................ Graduate Teaching/Research Associate, Department of Chemistry, The Ohio State University, Columbus, OH Publication You, J., Wang, L., Saji, M., Olesik, S. V., et al., High-sensitivity TFA-free LC-MS for profiling histones. Proteomics 2011, 11, 3326-3334. Field of Study Major Field: Chemistry viii Table of Contents Abstract .................................................................................................................ii Dedication ............................................................................................................ v Acknowledgments ................................................................................................vi Vita .................................................................................................................... viii Table of Contents ..................................................................................................ii List of Tables ........................................................................................................ v List of Tables in Appendix A .................................................................................vi List of Figures ..................................................................................................... viii List of Figures in Appendix A ................................................................................xi Abbreviation List ................................................................................................. xiii 1 Introduction .................................................................................................... 1 1.1 Proteomics and basic proteomic technologies ........................................ 1 1.2 Separation of proteomic analytes by liquid chromatography ................... 4 1.3 Separation of proteins by SDS-PAGE ..................................................... 6 1.4 Western blot ............................................................................................ 7 1.5 Immunoprecipitation ................................................................................ 8 1.6 Mass spectrometry .................................................................................. 8 1.6.1 Ion sources used for proteomic analysis ........................................... 9 1.6.2 Mass analyzer ................................................................................. 11 1.6.3 Tandem mass spectrometry ........................................................... 15 1.6.4 Peptide fragmentation by collision-induced dissociation ................. 17 1.7 Research goals ..................................................................................... 19 2 High-sensitivity TFA-free LC-MS for profiling histones ................................ 23 2.1 Introduction ............................................................................................ 23 2.2 Experimental ......................................................................................... 25 2.2.1 Preparation of cell lines ................................................................... 25 2.2.2 Preparation of protein standards ..................................................... 26 2.2.3 Extraction of histones from bovine calf thymus ............................... 27 2.2.4 Extraction of histones from human cells ......................................... 28 2.2.5 Liquid chromatography mass spectrometry (LC-MS) ...................... 28 ii 2.3 Results and discussion .......................................................................... 31 2.3.1 Effect of TFA ion-pairing agent on the ESI-MS spectra of histones 31 2.3.2 Optimization of TFA-free histone LC-MS ........................................ 36 2.3.3 TFA-free LC-MS analysis of protein standards ............................... 46 2.3.4 Application of TFA-free LC-MS analysis ......................................... 48 2.4 Conclusion ............................................................................................. 53 3 Targeted mass spectrometry
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