Protein post-translational modifications (PTMs), such as phosphorylation, glycosylation, oxidation and methylation, play critical roles in a variety of intra- and intercellular activities, such as cell growth, division, migration and apoptosis. Dysregulation of PTMs may induce various diseases including cancer and diabetes. Mass spectrometry (MS) based proteomics has gained popularity in identifying, characterizing and quantifying proteins with PTMs. Successful structural elucidation of proteins with PTMs largely replies on obtaining fragmentation information from tandem mass spectrometry (MS/MS) of peptides resulting from protein digestion. This dissertation partially focuses on improving the knowledge of phosphopeptide fragmentation chemistry during collision induced dissociation (CID)-MS/MS. Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of phosphopeptides. Two competing fragmentation pathways are involved in this process, namely the direct loss of H3PO4 and the combined losses of HPO3 and H2O. They produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS3 for phosphorylation site localization. Furthermore, phosphate group rearrangement reactions in CID-MS/MS (phosphate groups transfer from one phosphorylated site to another hydroxyl group in the peptide) increase the ambiguity for assigning phosphate groups. In this dissertation, factors influencing the competing fragmentation and phosphate group rearrangement reactions during CID-MS/MS of phosphopeptides have been systematically evaluated using a synthetic phosphopeptide library by varying a number of peptide properties. Both competing fragmentation and phosphate group rearrangement reactions were found to be most problematic for CID-MS/MS of phosphopeptide ions with limited proton mobility. The relative contribution of each competing neutral loss pathway was quantified in a series of regioselective 18O-phosphate ester labeled phosphopeptides by comparing the abundance of the -100 Da (-H3PO318O) versus -98 Da (-(HPO3+H2O)) neutral loss product ions formed upon CID-MS/MS.MS-based methods have also been extensively used for characterization and quantification of proteins containing methionine oxidation. Here parathyroid hormone (PTH), which is responsible for regulation of circulating calcium concentration in plasma, has been analyzed. The oxidation kinetics of PTH was first investigated in vitro with H2O2. The obtained oxidized forms of PTH were characterized by CID-MS/MS. It was found that methionine residues in PTH can mainly be oxidized to sulfoxides. An immuno-LC-MS/MS assay was then successfully developed for simultaneous quantification of native, truncated and oxidized forms of PTH in clinical plasma samples by employing heavy isotope labeled protein and peptide standards. The results from this immuno-LC-MS/MS assay analysis were compared to those from traditional PTH immunoassays to evaluate the effect of oxidation on the detection of PTH using traditional PTH immunoassays.
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