Novel methods and applications in quantitative mass spectrometry-based proteomics and lipidomics

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Advances in mass spectrometry (MS) instrumentation now make it possible to identify thousands of proteins or hundreds of lipids from a single biological sample. Despite this, in most experiments only a subset of the expected number of proteins or lipids are identified and reported in the literature. More recently, MS has emerged as a quantitative tool to monitor the dynamics of these biomolecules during the progression of biological events or in response to external stimuli. A number of MS-based quantitative tools have been developed with which to study proteins and lipids; however, few of these work on both proteins and lipids, many are cost prohibitive and many cause the sensitivity of the analyses to decrease. There is a need for new developments in MS-based quantitative protein and lipid analysis and this will continue to be the focus of the research program in the Smith laboratory. For this round of funding, efforts will be directed at further developing Trimethylation Enhancement using Diazomethane (TrEnDi). TrEnDi is a chemical derivatization technique we have developed that uses diazomethane to methylate protein and lipid functional groups that have pKa values lower than 11. In the case of peptides, this translates into the neutralization of negatively charged functional groups and the formation of fixed and permanent positive charges on the peptide. Our research will expand the utility of TrEnDi, using new chromatographic separation schemes to increase the sensitivity of peptide analysis, enabling regions of proteins normally invisible to MS-based methods to be observable. Methods using isotopically labelled diazomethane will be developed and will permit novel modes of quantitation at higher levels of sensitivity. Phosphopeptides will be modified by TrEnDi to facilitate their identification in complex samples in a novel and sensitive manner. In the case of lipids, TrEnDi increases the sensitivity of glycerophospholipids by neutralizing the negatively charged phosphate group and converting primary amines into quaternary amines. The major lipid constituents of cellular membranes, known to also play roles in cellular communication, will be modified by isotopically labelled diazomethane and analyzed by MS; preliminary results show TrEnDi increases the sensitivity for some lipid classes over 10 fold. HQP will drive the program in a highly collaborative environment and gain many attractive skill sets to the Canadian job market.