Development and Application of Top-down and Middle-down Proteomics for Comprehensive Protein Characterization in the Muscle Proteome
Author: Yutong Jin
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Published: 2019
Total Pages: 0
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DOWNLOAD EBOOKMass spectrometry (MS)-based proteomics, including top-down, middle-down and bottom-up approaches, has become the method of choice for protein identification, quantification, and characterization. Top-down proteomics represents a superior approach for comprehensive protein characterization by analyzing intact proteins, providing an overview of all the proteoforms present in a sample and allowing for identification of various proteoforms and in-depth characterization of sequences and post-translational modifications (PTMs). However, the protein mass range that can be analyzed by top-down approach is normally limited to 100 kDa. Middle-down proteomics, analyzing polypeptides from proteolysis, is a complementary strategy to top-down proteomics for characterization of large proteins (>100 kDa) and complex PTMs. In this dissertation, I developed and applied top-down and middle-down proteomics to comprehensively characterize the sarcomeric proteins from striated muscle tissues. Sarcomeric proteins are expressed in different isoforms produced by homologous genes, in the presence of various proteoforms produced by a single gene via genetic variations, alternative RNA splicing, and PTMs. The various isoforms and PTMs of sarcomeric proteins are associated with muscle contractile properties and thus affects the entire muscle function. However, a comprehensive characterization of sarcomeric protein isoforms and PTMs is still challenging due to the high complexity of the muscle proteome and the limitations in techniques for protein separation and characterization. The first part of this dissertation focuses on characterizing sarcomeric proteins below 100 kDa using top-down proteomics. Sarcomeric proteins from multiple skeletal muscle tissues were characterized by liquid chromatography and mass spectrometry plus (LC-MS+) strategy which combines online LC-MS and offline tandem MS (MS/MS) analysis. I also developed a reversed-phase chromatography method using monolithic column for highly efficient separation of sarcomeric proteins and combined it with high-resolution MS for top-down proteomics. The second part of this dissertation describes the development of middle-down proteomics for characterization of large sarcomeric proteins (>100 kDa). I developed a MS-compatible size-exclusion chromatography (SEC) method to purify myosin heavy chain from cardiac muscle tissue and characterize this protein by refining a middle-down MS strategy. I also developed a middle-down proteomics strategy to study the phosphorylation status of a serine-arginine-rich protein RNA-binding motif 20. Finally, combining top-down and middle-down proteomics, I described a strategy for the comprehensive characterization of a monoclonal antibody. Collectively, this dissertation provides comprehensive analysis of the isoforms and PTMs of sarcomeric proteins that will help elucidate molecular event in muscle contraction and disease pathologies. Furthermore, this dissertation presents a variety of novel LC and MS methods for protein separation and characterization, enabling the further characterization of other proteomes with deep proteome coverage.