MASS ANALYSIS OF PROTEINS UV CROSSLINKED TO NUCLEOTIDES

紫外交联至核苷酸的蛋白质的质量分析

• 批准号: 2906922
• 负责人: DOUGLAS F BAROFSKY
• 金额: $ 4.51万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2906922
• 关键词: DNA; Escherichia coli; adduct; bacterial proteins; bacteriophage T4; chemical binding; crosslink; electrospray ionization mass spectrometry; high performance liquid chromatography; matrix assisted laser desorption ionization; molecular site; nucleic acid sequence; photochemistry; transcription factor; ultraviolet radiation; virus protein

DESCRIPTION: (Adapted from the applicant's abstract) In general, this proposal is concerned with methods used to study interactions between proteins and nucleic acids. Elucidating these interactions is one of molecular biology's central goals and has become one of the most active pursuits in medical research. The specific aims of the proposed study are to develop a general experimental protocol for locating and characterizing the nucleic acid binding domains of proteins and to use that methodology to determine the nucleic acid binding sites on bacteriophage T4 gene 32 protein (gp32), on uracil-DNA glycosylase from Escherichia coli, and on transcription termination factor rho from Escherichia coli. The experimental approach to achieving these aims will be to fix the interaction between a protein and nucleic acid by means of ultraviolet (UV) light-induced photochemical crosslinking and then to locate and identify the particular amino acid and nucleotide residues that have been covalently bound to each other through a protocol that integrates high performance liquid chromatography (HPLC), matrix- assisted laser desorption ionization (MALDI) mass spectrometry, and electrospray-ionization (ESI) mass spectrometry. The advantages gained through combining these techniques should be substantial the speed, sensitivity, and specificity of the mass spectrometric and chromatographic techniques should increase both the number and types of biological experiments that can be conducted with the versatile photochemical crosslinking method. Collaboration with colleagues currently using UV crosslinking techniques to investigate protein- nucleic acid interactions will significantly increase the likelihood of success in this study. They will provide a molecular biological perspective that will be invaluable to the design, conduct and interpretation of the experiments described in this proposal, and they will supply samples that will assure results relevant to contemporary research. Their respective studies will benefit in return because, at every stage of development, the proposed methodology will be generating otherwise unobtainable structural information about the nucleic acid binding proteins they investigate.

描述：（改编自申请人的摘要）通常 提案与用于研究之间相互作用的方法有关 蛋白质和核酸。 阐明这些互动是 分子生物学的核心目标，并已成为最活跃的目标之一 医学研究的追求。 拟议研究的具体目的 是为定位和 表征蛋白质的核酸结合结构域并使用 确定核酸结合位点的方法 噬菌体T4基因32蛋白（GP32），从尿嘧啶-DNA糖基化酶上 大肠杆菌和转录终止因子Rho 大肠杆菌。 实现这些目标的实验方法将 通过固定蛋白质与核酸之间的相互作用 紫外线（UV）光化学交联，然后 找到并确定特定的氨基酸和核苷酸残基 通过一个协议，已经共价约束 整合高性能液相色谱法（HPLC），基质 - 辅助激光解吸电离（MALDI）质谱法和 电喷雾离子化（ESI）质谱法。 获得的优势 通过结合这些技术应该很高的速度， 质谱和特异性的敏感性和特异性 色谱技术应增加数量和类型 可以用多功能进行的生物实验 光化学交联方法。 与同事合作 目前使用紫外线交联技术研究蛋白质 核酸相互作用将显着增加 这项研究的成功。 他们将提供分子生物学 观点将对设计，行为和 对本提案中描述的实验的解释，他们 将提供将确保与当代相关的结果的样品 研究。 他们各自的研究将受益于回报 每个发展的阶段，拟议的方法将生成 否则关于核酸的无法获得的结构信息 他们研究的结合蛋白。

项目成果

Comparison of ESI-MS interfaces for the analysis of UV-crosslinked peptide-nucleic acid complexes.

用于分析 UV 交联肽-核酸复合物的 ESI-MS 接口比较。

• DOI: 10.1016/j.jchromb.2007.09.029
• 发表时间: 2007
• 期刊: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
• 作者: Gafken,PhilipR;Doneanu,CatalinE;Bennett,SamuelE;Barofsky,DouglasF
• 通讯作者: Barofsky,DouglasF