Structural Mapping Protein-Surface Interface by Proteomics

通过蛋白质组学绘制蛋白质-表面界面结构图

• 批准号: RGPIN-2017-06073
• 负责人: Sun, Bingyun
• 金额: $ 1.6万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=678890
• 关键词: Structural; Mapping; Protein; Surface; Interface

The ability to study proteins and membrane proteins in a small number of cells is critical to investigate stem-cell development and the cause of cancer. Proteome is the entire protein complement of a biological system. Proteomic study of a small number of stem cells and cancer initiating cells enables comprehensive characterization of protein functions in complex biological events that these cells participate in. The obtained knowledge can provide molecular guidance for disease treatment and prevention.*******Currently it is impossible to carry out membrane proteomics from fewer than 1000 cells, which is needed to address the above mentioned biological questions. One reason is the severe sample loss occurs in proteomic analysis of trace proteins (i.e. microproteomics). Aspecific adsorption of proteins not only hampers microproteomics, but also impacts a number of other research fields including biosensing, biomedicine, biodefense, as well as food, marine, and textile industries. Protein surface interactions are complex. A majority of proteins in the proteome have no structural information on how they interact with different solid surfaces; therefore, there is no effective strategy to prevent proteome-wise protein adsorption.*******CFI has awarded us a nanoflow liquid chromatography (LC) and accurate Fourier transform mass spectrometer (MS) coupled system for us to develop novel methodologies to investigate molecular structures at the protein-surface interface, and to analyze membrane proteome from a few stem cells. In the proposed research, we will use Chinese hamster ovary cells as a model and the awarded LC-MS system as the detection means to explore different chaotropes, surfactants, enzymes, and proteolytic methods for proteomic characterization of sub-microgram of protein adsorbed in regular sample vials. By tagging individual amino acids, we will probe the atomic interfacial structure for hundreds if not thousands of proteins. By modulating such interaction, we hope to achieve the sensitive analysis of proteome and membrane proteome from a few cells.*******This study will enable us to utilize developed methods to investigate the cell population heterogeneity not only in stem cells that prohibits its clinical translation, but also in cancer tissues that causes fractional kills in current therapies and frequent relapse. The discovered biomarkers of different subtypes of stem cells will facilitate the production of high quality and quantity stem cells needed in clinics, and the discovered druggable targets will enable complete eradication of cancer. The developed methods will also benefit other sensitive proteomic studies such as early diagnosis and prevention of diseases. The strategies to control protein aspecific adsorption will shed light on other research fields mentioned above, and the students trained in our program will help the research and application in these fields in the future.******

研究少量细胞中的蛋白质和膜蛋白的能力对于研究干细胞发育和癌症原因至关重要。蛋白质组是生物系统的完整蛋白质补充物。对少量干细胞和癌症起始细胞进行蛋白质组学研究，能够全面表征这些细胞参与的复杂生物事件中的蛋白质功能。所获得的知识可以为疾病的治疗和预防提供分子指导。********目前不可能对少于1000个细胞进行膜蛋白质组学，这是解决上述生物学问题所需要的。原因之一是痕量蛋白质的蛋白质组学分析（即微蛋白质组学）中发生严重的样品损失。蛋白质的非特异性吸附不仅阻碍了微蛋白质组学，而且还影响了许多其他研究领域，包括生物传感、生物医学、生物防御以及食品、海洋和纺织工业。蛋白质表面相互作用是复杂的。蛋白质组中的大多数蛋白质没有关于它们如何与不同固体表面相互作用的结构信息；因此，没有有效的策略来防止蛋白质组蛋白吸附。********CFI 授予我们纳流液相色谱 (LC) 和精确的傅里叶变换质谱仪 (MS) 耦合系统，以便我们开发新的方法来研究蛋白质-表面界面的分子结构，并分析一些干细胞的膜蛋白质组。在本研究中，我们将以中国仓鼠卵巢细胞为模型，以获奖的LC-MS系统为检测手段，探索不同的离液剂、表面活性剂、酶和蛋白水解方法，对常规样品瓶中吸附的亚微克蛋白质进行蛋白质组学表征。通过标记单个氨基酸，我们将探测数百甚至数千种蛋白质的原子界面结构。通过调节这种相互作用，我们希望实现对少数细胞的蛋白质组和膜蛋白质组的灵敏分析。********这项研究将使我们能够利用已开发的方法来研究细胞群异质性，不仅在阻止其临床转化的干细胞中，而且在导致当前疗法中部分杀伤和频繁复发的癌症组织中。所发现的不同干细胞亚型的生物标志物将有助于生产临床所需的高质量和数量的干细胞，所发现的药物靶点将能够彻底根除癌症。开发的方法还将有利于其他敏感的蛋白质组学研究，例如疾病的早期诊断和预防。控制蛋白质非特异性吸附的策略将为上述其他研究领域带来启发，我们项目培养的学生将有助于这些领域未来的研究和应用。******