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A Thesis Entitled Identification of Proteins A Thesis Entitled Identification of Proteins from Lanthionine Ketimine Ethyl Ester (LKE)- treated and untreated Rat Glioma 2 (RG2) Cells using Proteomic Approaches by Siddhita Abhijeet Shirsat Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Chemistry _________________________________________ Dr. Dragan Isailovic, Committee Chair _________________________________________ Dr. Donald R. Ronning, Committee Member _________________________________________ Dr. Jon R. Kirchhoff, Committee Member _________________________________________ Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2016 Copyright 2016, Siddhita Abhijeet Shirsat This document is copyrighted material.Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Identification of Proteins from Lanthionine Ketimine Ethyl Ester (LKE)- treated and untreated Rat Glioma 2 (RG2) Cells using Proteomic Approaches by Siddhita Abhijeet Shirsat Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Chemistry The University of Toledo August 2016 Glioma is a type of tumor which originates in the glial cells that surround and support neurons in the brain. According to the Central Brain Tumor Registry of the United States (CBTRUS), 29% of all central nervous system tumors diagnosed are gliomas. Rat glioma models and cell lines reflect high-grade brain tumors. Proteomics is commonly used to discover novel glioma biomarkers and study drug-protein interactionsin rat glioma models and cell lines. Proteomic studies are important because they provide a wealth of information about biochemical properties of brain tumors, and assist in discovery and validation of glioma biomarkers and drug targets. Here, proteomic techniques were used to explore the differences in the proteomes of malignant rat glioma 2 (RG2) cells in the presence and the absence of a sulphur- containing drug, lanthionine ketimine ethyl ester (LKE). In 2010, Hensley et al. synthesized LK from 3-bromopyruvate and L-cysteine hydrochloride as described by Cavallini et al., and LKE was synthesized using L-cysteine ethyl ester. It was shown that LKE promotes neurite elongation in neurons at nanomolar concentrations, protects iii neurons against oxidative stress, and suppresses microglial activation. Recently, Hensley et al. have analyzed the expression of proteins in RG2 cells that were treated with LKE using mostly immunoblotting techniques. A few proteins that are involved in the process of autophagy (cellular recycling) in RG2 cells were influenced by LKE. The objective of this project was to apply proteomic techniques and mass spectrometry (MS) to further study the differences in the protein expression levels in LKE-treated and untreated RG2 cells. Electrophoretic methods (e.g., SDS-PAGE), chromatographic techniques (e.g., nano-HPLC), and MS (e.g., MALDI-MS/MS and ESI- MS/MS) were used to study proteins and phosphoproteins from RG2 cells. Using nano- HPLC-MALDI-MS/MS, 22 proteins were identified from LKE-treated RG2 cells, and 18 proteins were identified from untreated RG2 cells. The number of identified proteins was significantly improved using nano-HPLC-ESI-MS/MS; 1681 proteins were identified from LKE-treated RG2 cells and 1457 proteins were identified from untreated RG2 cells. Specifically, the protein vimentin, which was considered as a potential biomarker in glioma cell line U87 was identified in both LKE-treated and untreated RG2 cells. 53 kinases from untreated RG2 cells and 65 kinases from LKE-treated RG2 cells were identified, including serine/threonine protein kinase mTOR and ribosomal protein S6 kinase (p70S6K), which were previously related to the process of autophagy in RG2 cells by Hensley et al. Overall, the present research results provide a long list of glioma-related proteins that can be detected and identified in LKE-treated and untreated RG2 cells using proteomic technologies. Future studies will aim to quantify the effect of LKE on the expression of glioma-related proteins in RG2 cells using HPLC-MS/MS in order to further explore the mechanism of interaction of LKE with proteins in glial cells. iv Dedicated to my caring and supportive husband, my loving parents, and to my wonderful siblings v Acknowledgements First and foremost, I am thankful to my supervisor Dr. Dragan Isailovic for his constant support and guidance from initial to the final level. I want to thank Dr. Kenneth Hensley for providing me biological samples for my projects and his constructive advice and ideas. I would like to thank my committee members Dr. Ronning and Dr. Kirchhoff for their valuable advice. I would also like to thank Dr. Leif Hanson for giving me training on how to use the MALDI mass spectrometer. I would like to convey my special thanks to Prof. Kippenhan for guiding me to be a good teacher and helping me to become an organized person and, more importantly, a better teaching assistant. I would like to thank my all current and former group members for their help in the lab. A special note of thanks goes to Rachel Marvin. The completion of this project could not have been possible without her support. I cannot express enough thanks to Rachel for her continued support and encouragement throughout these 3 years. I appreciate her help and wish her all the best in her future endeavors. Last but not the least; I would like to thank my Pappa, Mummy, my in-laws, and the most important person in my life my husband and my siblings Rani, Siddhu, Priyanka, for everything they have done. Their unconditional love, constant support, and encouragement always made me more confident and gave me strength to keep moving through all challenging situations. vi Table of contents: Abstract……………………………………...………..…………………………………..iii Acknowledgments………………………………………………………………………..vi Table of Contents………………………………………………………………………..vii List of Tables……………………………………………………………………………...x List of Figures……………………………………………………………………………xi List of Abbreviations……………………………………………………………………xiii List of Symbols………………………………………………………………………….xv 1. Introduction 1.1 LKE: synthesis and neurological effects.……...………………………………1 1.2 Proteomics……………………………………………………………………..4 1.3 Mass spectrometry…………………………………………………………….5 1.4 Ion sources…………………………………………………………………….7 1.4.1 Matrix-assisted laser desorption ionization (MALDI)……...……....7 1.4.2 Electrospray ionization (ESI) and nanoelectrospray ionization (nano-ESI)………………………………….……………………………11 1.5 Mass analyzers……………………………………………………………….13 1.5.1 Time-of-flight mass analyzer………………………………….…...15 1.5.2 Quadrupole mass analyzer….……………………………………...17 vii 1.5.3 Ion trap mass analyzer……….…………………………………….19 1.5.4 Orbitrap mass analyzer……….……………………………………21 1.6. Separation of proteins and peptides………………………………………...23 1.6.1 Gel electrophoresis.………………………………………………..23 1.6.2 Nano-high performance liquid chromatography (nano-HPLC).…..25 1.6.3 Nano-HPLC fraction collector.…………………………………….26 1.7 Protein identification by peptide mass fingerprinting (PMF).……………….27 1.8 Tandem mass spectrometry………………………………………………….28 1.9 Western blotting.…………………………………………………………….29 1.10 Proteomic studies of glial tissues and cells………….…………………..…30 2. Materials and methodology 2.1 Materials and Instruments…………………………………………………....32 2.2 Methodology.………………………………………………………………...33 2.2.1 Sample preparation.………………………………………………..33 2.2.2 Separation of proteins by SDS-PAGE……………….…………….33 2.2.3 In-gel tryptic digestion and peptide mass fingerprinting (PMF)..…35 2.2.4 In-solution tryptic digestion..………………………………………36 2.2.5 Separation and identification of peptides and proteins by nano-HPLC-MALDI-MS and MS/MS.………………………………….37 2.2.6 Separation and identification of peptides and proteins by nano-HPLC-ESI-MS and MS/MS.………………………………………38 viii 3. Results and discussion 3.1 The identification and relative quantification of proteins and phosphoproteins from LKE-treated and untreated RG2 cells using SDS-PAGE, protein staining and MALDI-MS.……………………………………………………………………..40 3.2 The identification of proteins from LKE-treated and untreated RG2 cells using nano-HPLC-MALDI-MS/MS……………………………………………………43 3.3 The identification of proteins from LKE-treated and untreated RG2 cells using nano-HPLC-ESI-MS/MS…………...……………………………………………47 3.4 Glioma related proteins identified using both nano-HPLC-MALDI-MS/MS and nano-HPLC-ESI-MS/MS…………..………………………………………..50 4. Conclusion and future work..……………………………………………………...55 4.1 Conclusions…………………………………………………………………..55 4.2 Future work…………………………………………………………………..56 References……………………………………………………………………………….58 Appendices………………………………………………………………………………68 ix List of Tables 1.1 A comparison of common mass analyzers …………………………………………14 3.1 A list of selected proteins related to glioma and cell proliferation identified in untreated and LKE-treated RG2 cells using both nano-HPLC-MALDI and ESI-MS/MS ………...............................................................................................................................51 3.2 An additional list of selected proteins related to glioma and cell proliferation identified in untreated and LKE-treated RG2 cells using nano-LC-ESI-MS/MS……….52 B.1 A list of proteins identified from untreated RG2 cells in two replicateusing nano- HPLC-MALDI-MS/MS experiments ...…………………………………………………72 B.2 A list of proteins identified from LKE-treated RG2 cells in two replicatenano-HPLC MALDI-MS/MSexperiments………………………………………………....................73
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