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Application of Different Bioanalytical Workflows for Proteomics of Prostate Cancer Li Chen University of Tennessee Health Science Center

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Application of Different Bioanalytical Workflows for Proteomics of Prostate Cancer Li Chen University of Tennessee Health Science Center University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 12-2009 Application of Different Bioanalytical Workflows for Proteomics of Prostate Cancer Li Chen University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Chen, Li , "Application of Different Bioanalytical Workflows for Proteomics of Prostate Cancer" (2009). Theses and Dissertations (ETD). Paper 43. http://dx.doi.org/10.21007/etd.cghs.2009.0050. This Dissertation is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. Application of Different Bioanalytical Workflows for Proteomics of Prostate Cancer Document Type Dissertation Degree Name Doctor of Philosophy (PhD) Program Pharmaceutical Sciences Research Advisor Sarka Beranova-Giorgianni, Ph.D. Committee Dominic M. Desiderio, Ph.D. Ivan C. Gerling, Ph.D. Wei Li, Ph.D. Duane D. Miller, Ph.D. DOI 10.21007/etd.cghs.2009.0050 This dissertation is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/43 APPLICATION OF DIFFERENT BIOANALYTICAL WORKFLOWS FOR PROTEOMICS OF PROSTATE CANCER A Dissertation Presented for The Graduate Studies Council The University of Tennessee Health Science Center In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy From The University of Tennessee By Li Chen December 2009 Chapter 2 © 2009 by American Chemical Society. All other material © 2009 by Li Chen All rights reserved ii DEDICATION To my husband and son, who remind me daily of the joys of life; and to my parents and my brother for their everlasting love and support. iii ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest and most sincere appreciation to my advisor and mentor, Dr. Sarka Beranova-Giorgianni, for the training I received in her lab. Thanks for her supervision, advice and encouragement for the research projects. I also thank her for the time she spent guiding me writing, editing and revising the manuscripts. I would also like to thank the other members of my committee, Dr. Duane D. Miller, Dr. Ivan C. Gerling, Dr. Dominic Desiderio, and Dr. Wei Li for their invaluable suggestions, guidance, and assistance. I would like to especially acknowledge Dr. Jeffrey R. Gingrich and Ms. Tina Barrett for generously providing me with the human prostate cancer samples and LNCaP cell lines without which a major portion of this research would not have been possible. I must also thank Dr. Ram I. Mahato and Mr. Michael Danquah for establishing the cooperation research work and providing the mouse prostate tumor samples for comparative proteomic study. I am especially grateful to Dr. Nataliya I. Lenchik in Dr. Gerling’s group for her training and assistance in the gel image analysis package and Ingenuity software. I would also like to thank Ms. Margaret M. Jefferson for her help with gel imaging equipment. I especially thank all former and current members in Dr. Beranova-Giorgianni’s Lab, Dr. Francesco Giorgianni, Dr. Amira Wohabrebbi, Dr. Yingxin Zhao, Dr. Bin Fang and Ms. Dina Mohamed Fallog, for technical help, discussions and suggestions. My thanks are extended to the staff in the Departments of Pharmaceutical Sciences, including Brenda Thornton for her administrative help in committee meeting; and Faith B. Barcroft for her help in reagent purchasing and administrative work. Tremendous thanks to my husband, my parents, my brother and my son. Without their incredible love and support, I would not accomplish my graduate training. iv ABSTRACT In the current dissertation, we focused on the development and application of multiple mass spectrometry-based bioanalytical platforms for phosphoproteomic characterization in cell culture and clinical specimens of prostate cancer; and on the application of optimized methods to analysis of differential protein expression to reveal molecular mechanism of drug action in animal model of prostate cancer. Characterization of the phosphoproteome in prostate cancer Our study in phosphoprotein signatures on a large scale in prostate cancer focused on the LNCaP human prostate cancer cell line, and on human prostate cancer tissue. For the LNCaP prostate cancer cell line, we applied a combination of analytical platforms: (1) a novel in-gel isoelectric focusing (IEF) LC-MS/MS analytical platform; (2) a 2-DE based platform combined with phospho-specific staining. The in-gel IEF LC-MS/MS analytical methodology used in the study included separation of the LNCaP proteins by in-gel isoelectric focusing; digestion of the proteins with trypsin; enrichment of the digests for phosphopeptides with immobilized metal ion affinity chromatography (IMAC); analysis of the enriched digests by LC-MS/MS; and identification of the phosphorylated peptides/proteins through searches of the Swiss-Prot protein sequence database. With in-gel IEF based analytical platform, we have characterized over 600 different phosphorylation sites in 296 phosphoproteins in the LNCaP prostate cancer cell line. This panel of the LNCaP phosphoproteins was 3-fold larger than the panel obtained in our previous work, and is the largest phosphoprotein panel in prostate cancer reported to date. The phosphoproteins identified in this study belonged to various locations within the cell and were involved in various processes including cell differentiation, transcription regulation, and intercellular signal transduction. We also developed a 2-DE based platform, in combination with multiplexed staining and LC-MS/MS, for the identification of LNCaP phosphoproteins. In this study, we applied 2-DE as separation technique, Pro-QTM Diamond stain as phosphoprotein detection method, LC-MS/MS and database searches for protein identification to investigate the phosphoproteins in the LNCaP prostate cancer cell line. Proteins identified from spots of interest were shown to be highly relevant to prostate cancer. We demonstrated the feasibility of using 2-DE with phospho-specific stain and mass spectrometry to investigate the phosphoproteins in the LNCaP cell line. This methodology complements the in-gel IEF LC-MS/MS platform that we used for phosphoproteomics study; it will be of particular value for future comparative studies of phosphoproteins in various physiological states. For prostate cancer tissue, a gel-free approach was performed to analyze five prostate cancer tissue specimens to obtain phosphoproteomic signature of prostate cancer v for biomarker discovery. Proteins were extracted with Trizol reagent, and then in-solution digested with trypsin. Phosphopeptides were enriched with IMAC, and analyzed the phosphorylated peptides/proteins by LC-MS/MS with identification through searches of the Swiss-Prot protein sequence database. The panels obtained for prostate cancer tissue contain 15-24 phosphoproteins. Some of the characterized phosphoproteins were present in all five specimens; in addition, each specimen also produced a unique set of phosphoproteins. The findings provided a direct glimpse into the phosphoprotein machinery operating within the human prostate cancer tissue. This pilot study focused on a small set of specimens. The phosphoprotein panels that were obtained contained a number of proteins that were unique to a particular specimen. Comparative proteomics study of drug effects in prostate cancer We carried out the first comparative proteomics study for the examination of the effects of bicalutamide/embelin combination treatment on prostate tumors by characterizing the alterations in protein expression that was induced upon treatment of mice bearing prostate tumors with anticancer combination therapy. A comparative proteomic strategy based on 2-DE coupled with LC-MS/MS was performed on mouse prostate tumor tissue. Proteins from the mouse prostate tumors were extracted with Trizol, and the protein mixtures were separated by 2-DE. Differences in the protein profiles for the different treatment groups were evaluated by computer- assisted analysis of SYPRO Ruby stained 2-DE gels. LC-MS/MS and database searches were used to identify differentially expressed proteins. Pathway analysis was carried out on the dataset of identified proteins with the Ingenuity bioinformatics tool. Out of the 33 differentially expressed protein spots, 30 protein spots were identified and grouped into various functional classes. The major protein categories were metabolism (52%), stress response (12%), protein biosynthesis (13%) and apoptosis (11%), suggesting that alterations in these processes may be involved in the mechanism of drug action. Proteins associated with oxidative stress were up-regulated, which indicated that treatment with bicalutamide/embelin may affect the redox balance within the prostate tumor, and this effect may contribute to tumor suppression. vi TABLE OF CONTENTS CHAPTER 1. INTRODUCTION................................................................................. 1 1.1 Prostate Cancer............................................................................................... 1 1.2 Why Apply Proteomic Technologies for Biomarker Discovery in Prostate Cancer? ..........................................................................................................
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