| Mass spectrometry is now well-established as a powerful tool for protein and peptide analysis, possessing both high sensitivity—with the ability to detect sequence-informative signals from as little as 50 attomoles of material—and great specificity—a single species can be isolated from a background of thousands of co-existing species.; The techniques of nano-flow RP-HPLC and micro-electrospray quadrupole ion trap mass spectrometry have been applied to the study of three biological systems. In the first, gel electrophoresis was used to resolve proteins co-immunoprecipitating with the Aurora B mitotic kinase from Xenopus laevis egg extracts. Aurora B plays critical regulatory roles in cell division, and has been implicated in the development of cancer in humans. Proteolysis in situ with trypsin, followed by mass spectrometry, allowed the identification of both known and novel molecular partners of Aurora B. A novel interaction between Aurora B and dynactin was discovered and verified biochemically.; In the second study, a molecular sequestration technique was employed to isolate substrates of the Escherichia coli proteolytic complexes ClpAP and ClpXP. These proteases are primary routes of bacterial protein degradation, targeting unfolded or missynthesized proteins and regulating cellular levels of specific substrates. We have identified several candidate substrates of ClpXP and ClpAP, and provide preliminary evidence for a possible motif for substrate recognition by the ClpXP protease.; In the third study, methodology is described for sequencing and identification of the repertoire of peptides presented on B cells by the class I major histocompatibility complex molecule HLA-A*0201. The class I MHC proteins display to the cellular immune system peptide fragments of expressed proteins. This provides protection against intracellular pathogens. We have employed multiple rounds of RP-HPLC fractionation and HPLC/MS to create a database of 685 HLA-A*0201-associated peptides. These peptides have been analyzed from both structural and cellular standpoints. Peptide structural features are described in the context of the crystal structure of HLA-A*0201, and cellular features are also discussed.; In the current work, mass spectrometry has been employed for peptide sequencing and protein identification in three diverse systems, demonstrating the value of mass spectrometry as a tool for discovery. |
