Acquisition of an Electrospray Mass Spectrometer

我们建议购买电喷雾HPLC质谱仪（单四极杆）进行四个不同的结构功能研究，为研究生和博士后研究员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量测定方法，该技术目前在校园内不可用。 第一个主要项目集中在细菌和哺乳动物DNA甲基转移酶。目标细菌酶是EcoRI DNA甲基转移酶。正在进行的这项NSF支持项目的工作使用了各种LC-MS方法，拟议的工作扩展了这一努力。提出了酶中关键组氨酸的表征;正在开发一种新型电喷雾LC-MS方法，并将其应用于甲基转移酶，以确定必需的组氨酸。提出的LC-MS方法是一个显着的改进，目前使用的分光光度法，可普遍适用。 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件与其底物接触。提出了一种相关的策略，以确定由酶接触的DNA底物的部分。还提出通过LC-MS方法研究哺乳动物酶的翻译后修饰的程度和位点。 哺乳动物酶的表征对于理解这种蛋白质在基因调控、癌症和遗传印记中起什么作用是重要的。 第二个主要项目集中在突触的乙酰胆碱转运蛋白 囊泡建议使用放射性标记的光亲和类似物和LC-MS方法鉴定蛋白质内的乙酰胆碱结合位点。 提出了转运蛋白的各种定点突变体的表征，包括这些突变体在其翻译后修饰中被修饰的程度。设计了一组相关的拟议实验，以评估不同的生长条件如何影响转运蛋白的翻译后加工。 拟议的结构功能分析的转运蛋白应有助于阐明如何神经递质乙酰胆碱的功能。 第三个项目建议使用LC-MS方法来阐明金属离子结合选择性的铁载体所产生的海洋细菌。最近确定的铁载体Alterobactin A，第一个结构特征的铁载体从开放的海洋生物的金属结合亲和力和选择性，将被确定。从而确定海水中铁载体的形态。提出了一种新的LC-MS方法来筛选大量潜在的金属;提出了其他海洋铁载体的应用。 第四个项目的重点是使用高场核磁共振。 研究蛋白质-核酸和蛋白质-蛋白质相互作用。提出LC-MS作为表征目标生物分子的附加手段。 购买拟议的LC-MS仪器将能够培训参与这些项目的许多研究生和研究员，以及本科生和与预期的少数用户有关的其他个人。这一拟议的培训针对生物技术行业对接受过现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.