Acquisition of an Electrospray Mass Spectrometer

我们建议购买一台电喷雾高效液相色谱质谱仪（单四极杆）来进行四种不同的结构-功能研究，为研究生和博士后提供新兴分析方法的培训，并为化学系和UCSB校园内的其他研究人员提供当代分子质量测定方法，这是目前校园内无法使用的技术。第一个主要项目侧重于细菌和哺乳动物DNA甲基转移酶。目标细菌酶是EcoRI DNA甲基转移酶。在这个NSF支持的项目中，正在进行的工作已经使用了各种LC-MS方法，拟议的工作扩展了这一努力。提出了酶内关键组氨酸的表征；一种新的电喷雾LC-MS方法正在开发中，应用于甲基转移酶应该能识别必需组氨酸。所提出的LC-MS方法是对目前使用的分光光度法的重大改进，可以普遍适用。提出了一种基于LC-MS的策略来确定哺乳动物胞嘧啶DNA甲基转移酶的哪些元素与其底物接触。提出了一种相关的策略来鉴定与酶接触的DNA底物的部分。该酶的翻译后修饰的程度和位点也建议用LC-MS方法进行研究。哺乳动物酶的特性对于理解这种蛋白质在基因调控、癌症和遗传印记中所起的作用是很重要的。第二个主要项目集中于突触囊泡的乙酰胆碱转运体。建议使用放射性标记光亲和类似物和LC-MS方法鉴定蛋白质内的乙酰胆碱结合位点。提出了转运体的各种位点定向突变体的特征，包括这些突变体在其翻译后修饰中的修饰程度。一组相关的实验被设计用来评估不同的生长条件对转运蛋白翻译后加工的影响。提出的转运蛋白的结构-功能分析应该有助于阐明神经递质乙酰胆碱的功能。第三个项目提出利用LC-MS方法研究开放海洋细菌产生的铁载体的金属离子结合选择性。将确定最近鉴定的铁载体Alterobactin A的金属结合亲和力和选择性，这是开放海洋生物中第一个结构表征的铁载体。从而可以确定海水中铁载体的形态。提出了一种新的LC-MS方法来筛选大量的潜在金属；提出了在其他海洋铁载体上的应用。第四个项目的重点是利用高场核磁共振来研究蛋白质-核酸和蛋白质-蛋白质相互作用。LC-MS被提议作为表征目标生物分子的另一种手段。购买拟议的LC-MS仪器将能够培训参与这些项目的众多研究生和研究员，以及本科生和其他与预期的未成年用户相关的个人。这项拟议的培训针对的是生物技术行业对接受当代分析方法培训的蛋白质化学家日益增长的需求。

We propose to purchase an electrospray HPLC mass spectrometer (single quadrupole) to carry out four diverse structure-function studies, to provide training in an emerging analytical method for graduate students and post doctoral fellows, and to provide other researchers within the Chemistry Department and UCSB campus access to a contemporary molecular mass determination method, a technique presently unavailable on the campus. The first major project focuses on both bacterial and mammalian DNA methyltransferases. The target bacterial enzyme is the EcoRI DNA methyltransferase. Ongoing work on this NSF supported project has used various LC-MS methods, and the proposed work extends this effort. Characterization of critical histidines within the enzyme is proposed; a novel electrospray LC-MS method is under development and application to the methyltransferase should identify the essential histidine(s). The proposed LC-MS method is a significant improvement over presently used spectrophotometric methods and may be generally applicable. An LC-MS based strategy is proposed to determine which elements of the mammalian cytosine DNA methyltransferase are in contact with its substrate. A related strategy is proposed to identify portions of the DNA substrate contacted by the enzyme. The extent and site of post- translational modification of the mammalian enzyme are also proposed to be investigated by LC-MS methods. The characterization of the mammalian enzyme is important for understanding what role this protein plays in gene regulation, cancer, and genetic imprinting. The second major project focuses on the acetylcholine transporter of synaptic vesicles. Identification of the acetylcholine binding site within the protein is proposed using radiolabeled photoaffinity analogs and LC-MS methods. Characterization of various site directed mutants of the transporter is proposed, including the extent to which these mutants are modified in their post-trans lational modification. A related proposed set of experiments is designed to assess how different growth conditions impact on the post-translational processing of the transporter. The proposed structure-function analysis of the transporter should help elucidate how the neurotransmitter acetylcholine functions. The third project proposes to use LC-MS methods to elucidate metal ion binding selectivities of siderophores produced by open ocean marine bacteria. The metal binding affinity and selectivity of the recently identified siderophore Alterobactin A, the first structurally characterized siderophore from an open ocean organism, will be determined. Thus the speciation of the siderophore in seawater can be determined. A novel LC-MS method is proposed to screen a large number of potential metals; application to other marine siderophores is proposed. The fourth project focuses on the use of high field N.M.R. to investigate protein- nucleic acid and protein-protein interactions. LC-MS is proposed as an additional means of characterizing the target biomolecules. The purchase of the proposed LC-MS instrument would enable the training of numerous graduate students and research fellows involved in these projects, as well as undergraduates and other individuals associated with intended minor users. This proposed training targets the increasing need in the biotechnology industry for protein chemists trained in contemporary analytical methods.