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

质谱（MS）仪器的进步现在可以从单个生物样品中识别数千种蛋白质或数百种脂质。尽管如此，在大多数实验中，只有预期数量的蛋白质或脂质的一个子集被鉴定并在文献中报道。最近，MS已经成为一种定量工具，用于监测生物事件进展期间或响应外部刺激时这些生物分子的动态。已经开发了许多基于MS的定量工具来研究蛋白质和脂质;然而，这些工具中很少对蛋白质和脂质进行研究，许多是成本高昂的，并且许多导致分析的灵敏度降低。基于MS的定量蛋白质和脂质分析需要新的发展，这将继续成为史密斯实验室研究计划的重点。对于这一轮的资金，将努力进一步开发使用重氮甲烷（TrEnDi）的三甲基化增强。TrEnDi是我们开发的一种化学衍生技术，使用重氮甲烷甲基化pKa值低于11的蛋白质和脂质官能团。在肽的情况下，这转化为带负电荷的官能团的中和和在肽上形成固定和永久的正电荷。我们的研究将扩大TrEnDi的实用性，使用新的色谱分离方案来提高肽分析的灵敏度，使基于MS的方法通常不可见的蛋白质区域能够被观察到。将开发使用同位素标记重氮甲烷的方法，并将允许以更高灵敏度进行定量的新模式。磷酸肽将被TrEnDi修饰，以便于以新颖和灵敏的方式在复杂样品中识别它们。在脂质的情况下，TrEnDi通过中和带负电荷的磷酸基团并将伯胺转化为季胺来增加甘油磷脂的敏感性。已知细胞膜的主要脂质成分也在细胞通讯中发挥作用，将通过同位素标记的重氮甲烷进行修饰并通过MS进行分析;初步结果显示TrEnDi将某些脂质类的灵敏度提高了10倍以上。HQP将在高度协作的环境中推动该计划，并为加拿大就业市场提供许多有吸引力的技能。

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.