Studies of Protein Function by Liquid Chromatography-Mass Spectrometry
Declan Williams, Gwillym
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Complete genomes of many organisms have been recorded using high throughput nucleic acid sequencing; however the intricate functions of the gene products remain largely undiscovered. As the most prominent technology for polypeptide sequence determination and quantification, mass spectrometry is used extensively to address questions regarding the behaviours of proteins in the biological context. The capacity of mass spectrometers to measure covalent modifications of amino acids is also of great value in the investigation of biological processes since specific reactive sites within proteins modulate their activity. Integration of liquid chromatography into mass spectrometry platforms has greatly improved sensitivity and throughput and is of particular benefit in the analysis of highly complex polypeptide mixtures. Studies of the behaviours of individual intracellular proteins in bacterial, plant and metazoan systems employing liquid chromatography-mass spectrometry are described herein. The phosphorylation of individual residues and resulting alterations in function were determined in GraR and VraR, two antibiotic resistance factors in Staphylococcus aureus. Three phosphosites of starch branching enzyme IIb which appear to regulate starch biosynthesis in Zea mays as well as kinases for which these sites are putative substrates were identified. Regulation of the multifunctional protein beta-catenin by p38 mitogen activated protein kinase was explored. Gene products with affinity for the highly conserved multifunctional protein beta-catenin, including several not previously known to interact, were identified from Rattus norvegicus smooth muscle cells. The specificity of peptide ion mass and peptide fragment ion mass relative to instrumental mass accuracy and the consequence for tandem-mass spectrometry-based quantification are explored in a separate chapter. In silico comparison of peptide ion/product ion mass pairs calculated from the human proteome revealed a range of mass redundancy from high to low simulated mass accuracy. Product ions from a single peptide sequence differ in their tendencies toward mass redundancy however no correlation between size and sequence specificity was apparent. This dissertation illustrates research into protein function conducted on three types of mass spectrometers and demonstrates some effects of liquid chromatographic and mass spectrometric performance on proteomic studies.