Structural Characterization of Polypeptides, Protein Complexes, Oligosaccharides

描述（由申请人提供）：UCSF Sandler-Moore质谱（SMMS）核心设施建议购买Synapt HDMS仪器，以便应用离子迁移率分离（IMS）技术，实现对各种生物分子（例如肽、蛋白质、蛋白质复合物、低聚糖和低脂糖）的高特异性组织成像和高级结构研究。我们寻求资金购买Waters Synapt HDMS系统，该系统提供了目前市场上其他仪器无法比拟的独特功能组合。此次收购将有利于来自加州大学旧金山分校的研究人员，以及我们与之保持密切合作的邻近研究机构：旧金山退伍军人事务医疗中心、加州大学伯克利分校和巴克年龄研究所。我们的长期目标是扩大我们的核心设施提供的基于质谱的服务范围，包括两个新的领域：(1)天然状态下的大型生物分子及其非共价组合的质谱分析，(2)基于ms的高清晰度组织成像，以及(3)我们希望通过添加IMS质量分析仪提供的额外分离阶段来增强我们目前在复杂基质中中等大小分子（例如肽、脂质、低聚糖）结构表征的能力。此外，MS(e)技术允许在MS/MS实验中提高采样效率，使Synapt HDMS仪器成为我们设施常规执行的蛋白质组学研究的有吸引力的工具。生物分子天然状态的质谱分析补充了基于高阶结构表征的经典光学检测技术。天然质谱领域的具体目标包括实施已知的协议和开发新的方法，用于基于质谱的蛋白质复合物和蛋白质配体组合的组成、化学计量学、结构和碰撞截面的阐明。具有良好表征的异质蛋白复合物作为模型（例如，septin）的可用性将使我们能够在将其应用于本提案中概述的目标研究之前验证我们的方案。重要的是，我们将与加州大学伯克利分校的Evan Williams博士密切合作，他是气相离子化学领域的专家，也是该应用程序的合作研究者。第二组目标涉及我们计划建立一种强大的方法来分析组织活检中的小分子空间分布（< 4 kDa）。我们不仅要绘制地图，还要确定感兴趣的物种，鉴于Synapt HDMS在该领域的文档功能，这是一个现实的目标。最后，我们将解决在不同浓度的复杂混合物中存在的类似结构的物种的识别和表征的需要。为此，我们将利用IMS来方便分析具有重叠m/z值的小组分，包括结构异构体。总之，Synapt HDMS将支持与癌症、生殖和内分泌疾病以及细菌感染直接相关的各种基础和转化研究。

DESCRIPTION (provided by applicant): The UCSF Sandler-Moore Mass Spectrometry (SMMS) Core Facility proposes acquire a Synapt HDMS instrument in order to apply ion mobility separation (IMS) technology to enable high specificity tissue imaging and advanced structural studies on various classes of biomolecules, (e.g., peptides, proteins, protein complexes, oligosaccharides and lipooligosaccharides). We seek funding to purchase a Waters Synapt HDMS system which offers a unique combination of capabilities currently not matched by other instruments on the market. This acquisition will benefit investigators from the UCSF campus as well as our neighboring research institutions with whom we maintain close collaborations: the San Francisco Veterans Affairs Medical Center, UC Berkeley and the Buck Institute for Age Research. Our long-term objective is to broaden the scope of mass spectrometry-based services that our core facility offers to include two new areas: (1) mass spectrometry of large biomolecules in their native state and their non-covalent assemblages, (2) high definition MS-based tissue imaging, and (3) we want to enhance our current capabilities in structural characterization of medium size molecules (e.g., peptides, lipids, oligosaccharides) within complex matrices by adding an additional stage of separation afforded by an IMS mass analyzer. Furthermore, the MS(e) technology that allows for increased sampling efficiency in MS/MS experiments makes the Synapt HDMS instrument an attractive tool for proteomic studies that our facility routinely performs. Mass spectrometry of biomolecules in their native state complements the classical, optical detection-based techniques of higher order structure characterization. Specific aims in the area of native mass spectrometry include implementation of known protocols and development of new methods for MS-based elucidation of composition, stoichiometry, architecture and collision cross section of protein complexes and protein-ligand assemblages. Availability of well characterized heteromeric protein complexes as models (e.g., septins) will allow us to validate our protocols before applying them to target studies that are outlined in this proposal. Importantly, we will work closely with Dr. Evan Williams from UC Berkeley who is an expert in the field of gas-phase ion chemistry and a contributing Co-Investigator on this application. A second set of aims addresses our plans to establish a robust method for analysis of small molecule spatial distributions (< 4 kDa) in tissue biopsies. We will not only map but also identify the species of interest, a realistic goal in view of the documented capabilities of the Synapt HDMS in this area. Finally, we will address the need for identification and characterization of species of similar structures that are present in complex mixtures at variable concentrations. To this end, we will take advantage of IMS to facilitate analysis of minor components with overlapping m/z values, including structural isomers. In conclusion, the Synapt HDMS will support a variety of basic and translational research studies directly relevant to cancer, reproductive and endocrine disorders and bacterial infections. PUBLIC HEALTH RELEVANCE: Detailed characterization of the structure and function relationships among various components of a living cell is a foundation of any research aiming at understanding pathophysiology of disease, developing cures and offering preventive measures. Waters Synapt HDMS system offers a versatile combination of technologies that enable structural characterization of very large "molecular machines" as well as small molecules, all responsible for the maintenance of a well balanced biological system. We will use this instrumentation to support studies on cancer, reproductive and endocrine disorders and bacterial infections.