Proteomic Analysis Using FTICR/MS

使用 FTICR/MS 进行蛋白质组分析

• 批准号: 6719692
• 负责人: I JONATHAN AMSTER
• 金额: $ 33.26万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-22 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6719692
• 关键词: Methanobacteriaceae; chemical synthesis; high throughput technology; hydrogen; liquid chromatography; mass spectrometry; matrix assisted laser desorption ionization; method development; peptides; protein quantitation /detection; proteolysis; proteomics; radionuclides

DESCRIPTION (provided by applicant): The proposed research is directed toward the development of new methods for quantitatively analyzing changes in protein expression in biological systems. The proposed developments will provide the means to analyze complex mixtures of proteins much more rapidly than is currently possible. Present day methodologies rely on the separation of protein mixtures into their individual components, followed by analysis for the identification of the separated proteins. The proposed developments will allow all proteins in a mixture to be identified simultaneously, thus providing a substantial reduction in analysis time and effort. Mixtures of proteins will be enzymatically digested, and the resulting mixture of proteolytic peptides will be separated by liquid chromatography and analyzed by high-resolution mass spectrometry. Proteins in the original mixture will be identified from their proteolytic fragments by using the accurate mass data that is produced. Presently, only a small proportion of peptides can be assigned to proteins by using accurate mass measurement. Most of the proposed effort will be directed into developing methods to increase the proportion of peptides that can be assigned to their parent proteins. A method called "mass defect labeling" is proposed as a way to increase the specificity of the assignment. Several novel reagents are proposed for mass defect labeling, and will be synthesized as part of the project. These reagents are not only useful for aiding protein identification, but also can be used to perform quantitative proteomics. Additional experiments will explore the use of endogenous labeling with a stable isotope in concert with the use of mass defect labels to achieve high specificity in protein identification. Using these methods together, calculations show that for the analysis of a prokaryotic proteome, up to 95% of the peptides that are measured can be assigned to the protein from which they derive. The success of the proposed developments will have great impact in biological research, drug discovery, and medicine. The proposed efforts will be carried out by both graduate and undergraduate students, and will be beneficial for their scientific development. The students involved in this research will be exposed to state-of-the-art, high-resolution mass spectrometry. This will provide society with well-trained scientists in this key technological area.

描述（由申请人提供）：拟议的研究旨在开发定量分析生物系统中蛋白质表达变化的新方法。拟议的进展将提供比目前更快地分析复杂蛋白质混合物的方法。目前的方法依赖于将蛋白质混合物分离成各自的成分，然后进行分析以鉴定分离的蛋白质。拟议的进展将允许同时鉴定混合物中的所有蛋白质，从而大大减少分析时间和工作量。蛋白质混合物将被酶消化，所得的蛋白水解肽混合物将通过液相色谱法分离并通过高分辨率质谱法进行分析。通过使用产生的精确质量数据，可以从其蛋白水解片段中鉴定出原始混合物中的蛋白质。目前，只有一小部分肽可以通过使用精确的质量测量来分配给蛋白质。大部分提议的努力将致力于开发方法来增加可分配给其母体蛋白质的肽的比例。提出了一种称为“质量缺陷标记”的方法作为增加分配特异性的方法。提出了几种用于质量缺陷标记的新型试剂，并将作为该项目的一部分进行合成。这些试剂不仅可用于帮助蛋白质鉴定，还可用于进行定量蛋白质组学。其他实验将探索使用稳定同位素内源标记与质量缺陷标记的使用相结合，以实现蛋白质鉴定的高特异性。结合使用这些方法，计算表明，对于原核蛋白质组的分析，高达 95% 的测量肽可以分配给它们所衍生的蛋白质。拟议开发的成功将对生物研究、药物发现和医学产生巨大影响。建议的努力将由研究生和本科生共同开展，将有利于他们的科学发展。参与这项研究的学生将接触到最先进的高分辨率质谱分析技术。这将为社会提供这一关键技术领域训练有素的科学家。