Development of Methodologies for the Proteoform-centric Analysis of Complex Proteomes
Proteoforms are the functional units of the cell, and their identification, characterization, and quantification are crucial for a comprehensive understanding of molecular processes in living systems. In this thesis, various approaches for the proteoform-centric analysis of complex proteomes were developed, including mass spectrometry (MS)-based bottom-up (BU), middle-down (MD), and top-down (TD) proteomics. In BU proteomics, proteoforms are enzymatically digested into peptides, which are identified and then assigned to protein groups by inference algorithms. MD proteomics is based on the same principle; however, larger peptides are generated during enzymatic digestion or chemical cleavage of the proteoforms. By contrast, TD proteomics aims to identify intact proteoforms. A BU proteomics approach using multiple proteases was developed, resulting in high protein sequence coverage providing strong evidence of truncated proteoforms. Moreover, a multi-dimensional MD proteomics strategy was designed, leading to more proteoform information than BU proteomics due to the identification of co-occurring modifications. Although BU and MD proteomics identified protein groups in great depth, elucidating proteoforms is inherently challenging due to the analysis of peptides and the need for protein inference. Thus, the main focus of the thesis was to develop and optimize methodologies for analyzing intact proteoforms by TD proteomics. Several proteoform fractionation strategies were established to decrease the sample complexity prior to mass spectrometric detection and increase the proteome coverage. The developed methodologies expand the toolbox of proteoform-centric analytics, demonstrating the potential and limitations of MS-based BU, MD, and TD proteomics. The various approaches facilitate the implementation of future TD proteomics experiments, which can contribute to the elucidation of molecular processes through the direct identification of proteoforms.
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