Studies of protein covalent modifications using mass spectrometry

Post-translational modifications (PTMs) of proteins are critical to protein functions. Furthermore, protein derivatization is becoming even more important in the study of the protein structure. During the past decade, the identification and characterization of protein covalent modifications have been driven by advances in mass spectrometry, which has been the focus of this dissertation.; Many PTMs are closely involved in the development of human diseases. For example, the cardiac troponin phosphorylation has been shown to be related to cardiac hypertrophy and heart failure. It is important to identify the exact phosphorylation sites to understand the physiological and pathological significance of troponin phosphorylation. With the application of THAP as the matrix for matrix assisted laser desorption/ionization (MALDI), post-source decay (PSD), and the immobilized metal ion affinity chromatograph (IMAC) technique, cardiac troponin phosphorylation was investigated.; Among the protein derivatization applications, the modification at cysteine residues is an important technique to study the protein structure using mass spectrometry because the sulfhydryl group of cysteine is highly reactive. We developed a biotinylation-tags-based method to successfully identify the most reactive free thiol group in glutathione-S-transferase (GST), which is important to understand the inhibition mechanism of this phase II metabolism enzyme via cysteine alkylation.; Cysteine derivatization and mass spectrometry were also used to investigate the structural difference between wild type and variant form of catechol O-methyltransferase (COMT). The peptide mass mapping results show that the variant form of COMT has a less stable global structure and is more sensitive to alkylation than wild type COMT. This result might explain why women with variant COMT are more likely to develop breast cancer.; A quantitative protein complex profiling method, termed as mass-coded abundance tagging (MCAT) was developed using guanidination of lysine residues. However, modification of the original MCAT method was necessary, because the ion intensities for the guanidinated and underivatized peptides species are probably different. A modified MCAT method was developed and then applied to the standard protein mixtures to prove that more accurate quantitation could be achieved than using the original MCAT procedure. The modified MCAT method was also applied to the study of protein expression changes in human prostate cancer cells caused by lycopene treatment.; In conclusion, new mass spectrometry based methods were developed to enhance the ability of mass spectrometry for peptide mapping of phosphorylated proteins, for studying the reactivities of cysteine residues in proteins, and for quantitative proteomics using guanidination of lysine residues. These methods were applied to investigate a variety of different protein covalent modifications. And these mass spectrometry based methods are applicable for other protein modification studies.