Bioanalytical applications of liquid chromatography mass spectrometry
Johnson, Jeremi David
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The goal of conventional proteomics is typically to identify, in a rapid and efficient manner, as many proteins within a particular extract as possible. Whole protein extracts can contain more than 10,000 proteins. The dynamic range of proteins in a whole protein extract presents but one daunting challenge. Other complications include a wide range of post-translational modifications, the potential for hundreds and even thousands of proteins, as well as the presence of detergents and other chemicals used in the extraction process. As a result, proteome analysis can become exceptionally challenging. Presented here are experiments where only a few proteins within a whole protein extract were of interest. The goal was to identify only those proteins associated with allergic responses. Post translational modifications (PTMs) are integral parts of many proteins. These modifications often manifest themselves in the form of N-linked glycosylation, O-linked glycosylation, and phosphorylation. A thorough understanding of protein structure and function encompasses the complete structural elucidation and localization of all PTMs. A variety of techniques including mass spectrometry, gel electrophoresis, antibody specific interactions, and targeted labeling, were utilized to tentatively solve all post translational modifications associated with several recombinant Pectate lyases. ESI-LC-MS and MALDI-MS were utilized to study the effects of point mutations on the specificity of a recombinant pectin methyl esterase. Successful characterization depended on the tandem mass spectrometric sequencing of a methyl esterified hexagalacturonate substrate. Utilizing a series of enzymes mutated to eliminate selected N-linked glycans, the role of N-linked glycosylation was assessed with regards to the enzyme’s ability to demethylate the aforementioned substrate. Unique to the substrate were the six potential sites available for enzyme catalyzed de-methylation. This allowed for assessment of the mode-of-action demonstrated by the mutated and wildtype enzymes.