Analytical Chemistry of Proteins and Peptides

蛋白质和肽的分析化学

• 批准号: RGPIN-2020-04677
• 负责人: Siu, KWMichael
• 金额: $ 2.62万
• 依托单位: University of Windsor
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717248
• 关键词: Analytical; Chemistry; Proteins; Peptides

Advances in proteomics have been made possible by conceptual developments and technological breakthroughs in biological mass spectrometry (MS). The long-term objective of my Discovery Grant (DG) research program aims at a better understanding of the fundamental aspects of the science that underpins peptide and protein identification and characterization, as well as developing analytical technologies that are critical to this goal. Within the next DG period, we will explore the structure, formation, energetics and fragmentation chemistries of (1) protonated peptides ([M + H]+); (2) molecular radical cations of peptides (M+); and (3) metallated peptide complexes by means of a combination of experimental and theoretical approaches. Additionally, we aim at developing methods to detect, characterize, and quantify phosphorylated proteins and peptides. Experimental techniques will include: selective enrichment and liquid chromatography (LC), tandem MS with isotopic labeling, and infrared multiple photon dissociation (IRMPD) spectroscopy. To augment structural and mechanistic determinations, density functional theory (DFT) calculations will be employed. We will investigate the loss of water from [M + H]+; for oligopeptides comprising only alkyl residues, the source is the peptide bonds, which lose water competitively to give isomers of protonated imidazol-4-ones that can interconvert or dissociate independently. The effect of the residue side chain will be examined. We will also investigate the loss of water from M+, especially in peptides in which presence of the radical is apparently critical to water loss. Side-chain effects of residues will also be examined. We will fully explore the effects of lanthanides (Ln's), including Eu3+ and Ce3+, as the ionization agents in metallated peptides. Preliminary data indicate that Eu3+ and Ce3+ complexes exhibit very different chemistries, strongly suggesting different binding modes. We will undertake a new, major initiative in our DG research on phosphorylated peptides. We will determine by both theory and experiment the peptide structures which favour intramolecular phosphate migration (scrambling) under MS conditions. We will develop analytical technologies to enable selective enrichment of phosphopeptides for protein characterization as well as for determination of phosphoproteomes of cancers. Preliminary investigations have yielded a Zr4+-chelated adenosine triphosphate (ATP)-functionalized silica monolith that exhibited superior sensitivity and selectivity against -casein, a model phosphoprotein, which resulted in the observation of 19 out of a total of 20 known phosphosites and a detection limit of 1 fmol. We will develop the metal-ion-chelated ATP-functionalized silica monolith column in tandem with nanobore reverse-phase LC for determination of the phosphoproteomes of oral cancers and precancers that we have examined and for which biomarkers have been identified and commercialized.

生物质谱（MS）的概念发展和技术突破使蛋白质组学的进步成为可能。我的发现基金（DG）研究项目的长期目标是更好地理解支撑肽和蛋白质鉴定和表征的科学的基本方面，以及开发对这一目标至关重要的分析技术。在下一节课中，我们将探讨(1)质子化肽（[M + H]+）的结构、形成、能量学和碎片化化学；(2)多肽的分子自由基阳离子（M+）；(3)采用实验与理论相结合的方法制备金属化肽配合物。此外，我们的目标是开发检测，表征和量化磷酸化蛋白和肽的方法。实验技术将包括：选择性富集和液相色谱（LC），串联质谱与同位素标记，红外多光子解离（IRMPD）光谱。为了增加结构和机械的决定，密度泛函理论（DFT）计算将被采用。