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

现在，质谱学(MS)仪器的进步使得从单个生物样本中识别数千种蛋白质或数百种脂类成为可能。尽管如此，在大多数实验中，文献中只鉴定和报告了预期数量的蛋白质或脂肪的子集。最近，MS已经作为一种定量工具出现，用于监测这些生物分子在生物事件进展过程中或对外部刺激的反应中的动态。已经开发了一些基于MS的定量工具来研究蛋白质和脂类；然而，这些工具很少同时用于蛋白质和脂类，许多工具的成本过高，许多工具导致分析的灵敏度降低。基于MS的定量蛋白质和脂肪分析需要新的发展，这将继续是史密斯实验室研究计划的重点。对于这一轮资金，将努力进一步开发使用重氮甲烷(Trendi)的三甲基化强化技术。Trendi是我们开发的一种化学衍生化技术，它使用重氮甲烷将pKa值低于11的蛋白质和脂肪官能团甲基化。对于多肽来说，这转化为带负电荷的官能团的中和和在多肽上形成固定和永久的正电荷。我们的研究将扩大Trendi的用途，使用新的层析分离方案来提高肽分析的灵敏度，使基于MS的方法通常看不到的蛋白质区域能够被观察到。将开发使用同位素标记的重氮甲烷的方法，并将允许以更高的灵敏度水平进行新的定量模式。Trendi将对磷酸肽进行修饰，以便于以一种新颖和敏感的方式在复杂样品中识别它们。在脂质方面，Trendi通过中和带负电荷的磷酸基团并将伯胺转化为季胺来提高甘油磷脂的敏感性。细胞膜的主要脂类成分，已知也在细胞通讯中发挥作用，将被同位素标记的重氮甲烷修饰并用MS进行分析；初步结果显示，Trendi将某些脂类的敏感度提高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获得许多有吸引力的技能集。

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.