Strategies for shotgun proteomics
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Shotgun proteomic assays utilizing peptide assignment by accurate mass analysis can increase sample throughput. This thesis describes two shotgun proteomics strategies to facilitate proteomics analysis by accurate mass measurement using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). ArgC digestion of a protein produces peptides that are more specific for protein identification than those from trypsinolysis. However, ArgC lacks specificity, displaying some trypsin-like behavior. This thesis reports a method for mimicking an ArgC digestion using lysine acetylation and trypsin digestion. Furthermore, it describes a novel technique for improving protein identification by isolating N-terminal peptides using a mass defect labeling approach to remove stray internal peptides. This approach to N-terminal peptide restricted shotgun proteomics provides a substantial gain in assignment specificity.