DECIPHERING THE DYNAMICS OF NEWLY SYNTHESIZED PROTEINS IN HUMAN CELLS USING MASS SPECTROMETRY-BASED PROTEOMICS
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Tong, Ming
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Abstract
Protein synthesis is extremely important for nearly every cellular activity. Newly synthesized proteins contain much valuable information about biological processes, and global analysis of these proteins and their dynamics can facilitate our understanding of cellular activities and the underlying mechanisms of diseases. However, it is extraordinarily challenging because of the low abundance of newly synthesized proteins among many highly abundant existing ones, the dynamic nature of these proteins, and the similarity between newly synthesized and preexisting mature proteins. The emergence of mass spectrometry (MS)-based proteomics has great potential for comprehensive analysis of newly synthesized proteins and their dynamics due to its high sensitivity, throughput, and accuracy.
In this thesis work, MS-based proteomics methods were applied to study the synthesis and degradation of newly synthesized proteins in human cells. An introduction of MS-based proteomics methods, including stable isotope labeling with amino acids in cell culture (SILAC)-based, azidohomoalanine (AHA)-based, puromycin-based, and cell-type-specific methods, for studies of newly synthesized proteins and their dynamics is provided in Chapter 1. Additionally, the applications of these methods and biological findings in the last several years are discussed. In Chapter 2, the degradation of newly synthesized proteins in human cells was comprehensively investigated by integrating metabolic labeling, click chemistry, and multiplexed proteomics. Systematic and quantitative analysis of newly synthesized proteins first revealed the degradation pathways of many proteins. Chapter 3 provides a new model considering the different degradation dynamics between the well-folded and misfolded forms of a protein to better describe the degradation of many proteins in cells. Chapter 4 describes a method to comprehensively evaluate the inhibition efficiencies of four commonly used inhibitors through global analysis of newly synthesized proteins. Chapter 5 focuses on the development of a novel mass spectrometry-based method termed quantitative O-propargyl-puromycin tagging (QOT) for accurate and sensitive investigation of rapid changes of protein synthesis in human cells by combining OPP labeling, click chemistry, and multiplexed proteomics. Finally, Chapter 6 summarizes the results of the thesis work and presents the outlook of the studies of newly synthesized proteins.
In conclusion, by integrating metabolic labeling and MS-based proteomics, newly synthesized proteins were systematically quantified to study the degradation pathways, protein folding and stability, the inhibition efficiencies of synthesis inhibitors, and the rapid changes of protein synthesis. New biological findings and methods provided in this thesis leads to a better understanding of biological processes and cellular metabolisms.
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2020-12-06
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Dissertation