UC Riverside

Mass spectrometry (MS), coupled with highly developed sample preparation and separation techniques, has served as one of the most powerful analytical technique. In this dissertation, we focus on the application of MS in two important areas, quantitative proteomics and protein post-translation modification (PTM) analysis.

In Chapters 2 and 3, we report the use of mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in HL-60 and K562 cells that were untreated or treated with a clinically relevant concentration of arsenite or imatinib. Our results revealed that, among more than 1000 quantified proteins, 56 and 73 proteins including many important enzymes had significantly altered levels of expression by arsenite and imatinib treatment, respectively. With the down-stream pathway analysis, our results provided potential biomarkers for monitoring the therapeutic intervention of leukemia and offered important new knowledge for gaining insight into the molecular mechanisms of action of anti-cancer drugs.

In Chapters 4 and 5, we obtained relatively comprehensive mappings of core histone post-translational modifications in two important model eukaryotic organisms, Neurospora crassa and Schizosaccharomyces pombe. We used several mass spectrometric techniques, coupled with HPLC separation and multiple protease digestion, to identify the methylation, acetylation and phosphoration sites in core histones. Our analysis provides potentially comprehensive pictures of core histones PTMs, which serve as foundations for future studies on the function of histone PTMs in these organisms.

In Chapter 6, we observed an unusual discrepancy between MALDI-MS/MS and ESI-MS/MS on the methylation of trimethyllysine-containing peptides. It turned out that the discrepancy could be attributed to an unusual methyl group migration from the side chain of trimethyllysine to the C-terminal arginine residue during peptide fragmentation. The results highlighted that caution should be exerted while MS/MS of singly charged ions is employed to interrogate the PTMs of trimethyllysine-containing peptides.