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

基于定量质谱的生物分子分析的新方法和应用

• 批准号: RGPIN-2020-06655
• 负责人: Smith, Jeffrey
• 金额: $ 2.11万
• 依托单位: Carleton University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741360
• 关键词: Novel; methods; applications; quantitative; mass

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.

质谱（MS）仪器的进步现在使得从单个生物样品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，文献中只识别并报告了预期数量的生物分子的一个子集。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生物分子的动力学。已经开发了许多基于MS的定量工具来研究生物分子，但是很少有人同时研究不同的生物分子类别。大多数是成本高昂的，许多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，这将继续成为史密斯实验室研究计划的重点。将努力使用重氮甲烷（TrEnDi）进行三甲基化增强的新创新。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的官能团。在肽、脂质和代谢物的情况下，这转化为带负电荷的官能团的中和以及固定和永久正电荷的形成，以增强电离并增加灵敏度。我们的研究建议开发一种全新的策略来进行TrEnDi，其中原位产生和使用重氮甲烷。这一发展将提高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学中受益。创建一个自动化平台来进行原位TrEnDi（iTrEnDi）将允许对化学进行更好的控制，并实现高通量分析。采用iTrEnDi以增强的灵敏度水平鉴定磷酸肽、甲基化肽和甘油磷脂的新方法将被开发，从而比以前更深入地了解细胞生物化学。iTrEnDi还将首次用于对源自体外的生物液体中的小分子进行化学衍生。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原因。我们的努力将通过在更低浓度下检测这些药物来改善法医和医学分析。最后，我们将使用iTrEnDi来增强草甘膦的分析特性，草甘膦是有争议的除草剂Roundup中的活性成分。我们希望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。