DISSERTATION INVESTIGATION OF MOLECULAR EFFECTS OF THE SOY-DERIVED PHYTOESTROGEN GENISTEIN ON CARDIOMYOCYTES BY PROTEOMIC ANALYSIS Submitted by Zeyu Sun Department of Chemical and Biological Engineering In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Fall 2011 Doctoral Committee: Advisor: Kenneth Reardon Co-Advisor: Karyn Hamilton Christopher Orton Bradley Reisfeld ABSTRACT INVESTIGATION OF MOLECULAR EFFECTS OF THE SOY-DERIVED PHYTOESTROGEN GENISTEIN ON CARDIOMYOCYTES BY PROTEOMIC ANALYSIS The soy-derived phytoestrogen genistein (GEN) has received attention for its potential to benefit the cardiovascular system by providing protection to cardiomyocytes against pathophysiological stresses. Although GEN is a well-known estrogen receptor (ER) agonist and a non-specific tyrosine kinase inhibitor, current understanding of the complex cellular and molecular effects of GEN in cardiomyocytes is still incomplete. The overall goal of this dissertation is to use high throughput proteomics methodologies to better understand the molecular action of GEN in cardiomyocytes and to identify proteins and pathways that respond to GEN treatment. The first study of this project focused on the concentration-dependent proteome changes in cultured HL-1 cardiomyocytes due to GEN treatments. Proteins from HL-1 cardiomyocytes treated with 1 µM and 50 µM GEN were prefractionated into hydrophilic and hydrophobic protein fractions and were analyzed by two-dimensional electrophoresis followed by protein identification using tandem mass spectrometry (MS). In total, 25 and 62 differential expressed proteins were identified in response to 1 µM and 50 µM of GEN treatment, respectively. These results suggest that 1 µM GEN enhanced the expression of heat shock proteins and anti-apoptotic proteins, while 50 µM GEN down-regulated glycolytic and antioxidant enzymes, potentially making cardiomyocytes more susceptible to energy depletion and apoptosis. The second study, employing a two-dimensional liquid chromatography and tandem MS shotgun proteomics workflow, was carried out to dissect the cellular functions changed in cardiomyocytes by ER-dependent or ER-independent actions of GEN. In this study, ii primary cardiomyocytes isolated from male adult SD rats were treated with 10 µM GEN without or with 10 µM ER antagonist ICI 182,780 (ERA) before proteomics comparison. A total of 14 and 15 proteins were found differentially expressed in response to the GEN, and the GEN+ERA treatment, respectively. Cellular functions such as glucose and fatty acid metabolism and cardioprotection were found to be modulated by GEN in an ER-dependent fashion, while proteins involved with steroidogenesis and estrogen signaling were identified as novel effectors of GEN via ER-independent actions. In this study, a consensus-iterative searching strategy was also developed to enhance the sensitivity of the shotgun proteomic approach. In the last study, an attempt to explore the response to a GEN stimulus in the signaling pathways, we developed a phosphopeptide enrichment method to assist the detection of protein phosphorylation in a complex peptide mixture. The quantitative performance of a sequential immobilized metal affinity chromatography (SIMAC) protocol was evaluated. We further conducted a preliminary application of this protocol in a large-scale, quantitative, label-free phosphoproteomics study to explore the alterations of protein phosphorylation patterns due to ER-independent GEN action in the SD rat cardiomyocytes. This project demonstrates the usefulness of proteomics methodologies to screen novel molecular targets influenced by GEN in cardiomyocytes. This is also the first investigation of the complex cellular impact of this soy-derived phytoestrogen in cardiomyocytes via a systems biology perspective. iii ACKNOWLEDGEMENT I would like to express my gratitude to all my committee members, Dr. Kenneth Reardon, Dr. Karyn Hamilton, Dr. Christopher Orton and Dr. Bradley Reisfeld who gave me the possibility to complete this dissertation and all the assistance along the journey. I owe my deepest gratitude to my advisor Dr. Reardon and co-advisor Dr. Hamilton for being extraordinary mentors for me by sharing their academic expertise and experience with me, and by providing guidance and encouragement throughout my Ph.D. work. It is an honor for me to work with them these years. I like to show my gratitude to all helping hands of all past and present members in Dr.Reardon’s lab and Dr. Hamilton’s lab. I also want to thank the following people who provided critical technical assistance and constructive advices: Dr. Carla Lacerda, Dr. Nichole Reisdorph, Dr. Jessica Prenni, Dr. Ann Hess, Dr. Andrey Ptitsyn, Dr. William Claycomb, Brian Cranmer, Delian Yang, Laurie Biela and Reuland Nellie. This work cannot be accomplished without their generous assistance and valuable input. Two outstanding undergraduate students have actively participated in my research: Kathryn Knopinski and Caitlin Mitchel. I want to use this opportunity to thank their hard working and their contributions to my research progress. Finally, I would like to say thank you to all my family and friends who have encouraged me during these years. Especially, I would like to give my special thanks to my mother Jiali Zhou, father Weiqiang Sun, and my fiancée Bing Chi for their patient support behind me to complete this work. Zeyu Sun At Dept. of Chemical and Biological Engineering Sep-11-2011 iv TABLE OF CONTENTS Chapter 1 Background and Objectives ......................................................................................... 1 1. Introduction ................................................................................................................. 1 1.1. Soy phytoestrogens and cardiovascular health .......................................................... 1 1.2. Cardiomyocytes and cardioprotection ...................................................................... 3 1.3. Genistein, chemical and biological properties ........................................................... 6 1.4. Proteomics and phosphoproteomics ....................................................................... 14 1.5. Models .................................................................................................................. 16 2. Objectives and Contributions of This Dissertation ...................................................... 18 Chapter 2 Phosphoproteomics and Molecular Cardiology: Techniques, Applications and Challenges ................................................................................................................................ 42 1. Introduction ............................................................................................................... 42 2. General Sample Preparation Strategies ....................................................................... 44 3. Subcellular Fractionation ........................................................................................... 45 4. 2DE Workflow .......................................................................................................... 47 4.1. Autoradiography .................................................................................................... 48 4.2. Phosphoprotein stains ............................................................................................ 49 4.3. Immunoblotting ..................................................................................................... 49 5. Liquid Chromatographic Methods .............................................................................. 50 5.1. Strong cation exchange liquid chromatography ...................................................... 52 5.2. Strong anion exchange liquid chromatography ....................................................... 53 5.3. Hydrophilic interaction liquid chromatography ...................................................... 54 5.4. Electrostatic repulsion hydrophilic interaction chromatography .............................. 55 6. Affinity Enrichment Strategies ................................................................................... 56 6.1. General considerations of using enrichment strategy .............................................. 56 6.2. Immunoaffinity method ......................................................................................... 58 6.3. Immobilized metal affinity chromatography ........................................................... 58 6.4. Metal oxide affinity chromatography ..................................................................... 60 6.5. Chemical derivatization methods ........................................................................... 61 7. Identification of Phosphopeptides by Tandem Mass Spectrometry .............................. 63 7.1. General considerations ........................................................................................... 63 7.2. Collision-induced dissociation with neutral loss scan ............................................. 64 7.3. Electron transfer dissociation ................................................................................. 66 8. Bioinformatics for Phosphoproteomics ...................................................................... 67 8.1. General procedure .................................................................................................. 67 8.2. Peptide identification ............................................................................................