Novel methods and applications in quantitative mass spectrometry-based biomolecule analysis

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个生物分子成为可能。尽管如此，在大多数实验中，文献中只识别和报告了预期数量的生物分子的子集。最近，MS已经作为一种定量工具出现，用于监测生物事件进展过程中或对外部刺激做出反应时生物分子的动力学。人们已经开发了一些基于MS的定量工具来研究生物分子，但很少有人同时研究不同的生物分子类别。大多数都是成本高昂的，而且许多会导致分析灵敏度降低。基于MS的定量生物分子分析需要新的发展，这将继续是史密斯实验室研究计划的重点。将致力于利用重氮甲烷(Trendi)进行三甲基化强化的新创新。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的官能团甲基化。对于肽、脂类和代谢物，这转化为带负电荷的官能团的中和和形成固定和永久的正电荷，以增强电离和提高灵敏度。我们的研究建议开发一种全新的策略来进行Trendi，其中重氮甲烷被创建并在现场使用。这一发展将增强Trendi的安全性，并使不同类型的分析物能够从这种强大的衍生化化学中受益。创建一个进行原位Trendi(ITrEnDi)的自动化平台将允许更好地控制化学并实现高通量分析。将开发利用iTrEnDi以更高的灵敏度识别磷肽、甲基化多肽和甘油磷脂的新方法，从而比以前更深入地了解细胞生物化学。ITrEnDi还将首次用于对来自体外的生物液中发现的小分子进行化学衍生化。我们的努力将集中在提高芬太尼和卡芬太尼的可检测性，这两种致命的阿片类药物是我们在加拿大面临的阿片类药物危机的主要责任。我们的努力将通过检测更低浓度的这些药物来改进法医和医学分析。最后，我们将使用iTrEnDi来增强草甘膦的分析特性，草甘膦是备受争议的除草剂农达的有效成分。我们预计，我们的发展将使草甘膦和其他除草剂能够在比以前更低的浓度下被检测到，从而能够进行严格的环境和生物医学研究。HQP将在一个高度协作和包容的环境中推动研究计划，获得对加拿大就业市场有吸引力的技能集。

Advances in mass spectrometry (MS) instrumentation now make it possible to identify thousands of biomolecules from a single biological sample. Despite this, in most experiments only a subset of the expected number of biomolecules are identified and reported in the literature. More recently, MS has emerged as a quantitative tool to monitor the dynamics of 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 biomolecules, however few work on different biomolecule classes at the same time. Most are cost prohibitive and many cause the analytical sensitivity to decrease. There is a need for new developments in MS-based quantitative biomolecule analysis and this will continue to be the focus of the research program in the Smith laboratory. Efforts will be directed toward new innovations using Trimethylation Enhancement using Diazomethane (TrEnDi). TrEnDi is a chemical derivatization technique we developed that uses diazomethane to methylate functional groups that have pKa values lower than 11. In the case of peptides, lipids and metabolites, this translates into the neutralization of negatively charged functional groups and the formation of fixed and permanent positive charges to enhance ionization and increase sensitivity. Our research proposes to develop an entirely new strategy to conduct TrEnDi where diazomethane is created and used in situ. This development will enhance the safety of TrEnDi and enable different types of analytes to benefit from this powerful derivatization chemistry. The creation of an automated platform to conduct in situ TrEnDi (iTrEnDi) will allow greater control over the chemistry and enable high throughput analyses. Novel methods employing iTrEnDi to identify phosphopeptides, methylated peptides and glycerophospholipids at enhanced levels of sensitivity will be developed opening the door to greater insight into cellular biochemistry than possible before. For the first time, iTrEnDi will also be used to chemically derivatize small molecules found in biological fluids that originate from outside the body. Our efforts will focus on enhancing the detectability of fentanyl and carfentanil, two deadly opioids that are primarily responsible for the opioid crisis that we are facing in Canada. Our efforts will improve forensic and medical analyses by detecting these drugs at even lower concentrations. Finally, we will use iTrEnDi to enhance the analytical characteristics of glyphosate, the active ingredient in the controversial weed killer Roundup. We expect that our developments will enable glyphosate and other herbicides to be detected at lower concentrations than previously possible enabling rigorous environmental and biomedical studies. HQP will drive the research program in a highly collaborative and inclusive environment gaining skill sets that are attractive to the Canadian job market.