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: $ 3.21万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748115
• 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的原因至关重要。蛋白质组是生物系统的全部蛋白质组成。对少量干细胞和癌症起始细胞的蛋白质组学研究使得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。目前不可能从少于1000个细胞中进行膜蛋白质组学，这需要解决上述生物学问题。一个原因是在痕量蛋白质的蛋白质组学分析（即微量蛋白质组学）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。蛋白质表面的相互作用是复杂的。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的蛋白质吸附。CFI授予我们一个纳米流液相色谱（LC）和精确的傅里叶变换质谱仪（MS）耦合系统，让我们开发新的方法来研究蛋白质-表面界面的分子结构，并分析了一些干细胞的膜蛋白质组。在拟议的研究中，我们将使用中国仓鼠卵巢细胞作为模型，并获得LC-MS系统作为检测手段，探索不同的离液剂，表面活性剂，酶和蛋白水解方法，用于常规样品小瓶中吸附的亚微克蛋白质的蛋白质组学表征。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，我们希望能够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先进的方法来研究细胞群体异质性，不仅在干细胞中，阻止其临床翻译，而且在癌症组织中，导致目前的治疗中的部分杀死和频繁复发。发现的不同干细胞亚型的生物标志物将促进临床所需的高质量和数量的干细胞的生产，并且发现的可药用靶点将能够完全根除癌症。该方法的发展也将有利于其他敏感蛋白质组学研究，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提供启示，我们培养的学生将有助于未来这些领域的研究和应用。