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新方法，用于定量分析生物系统中蛋白质表达的变化。拟议的开发将为分析蛋白质复杂混合物的速度比目前的可能性要快得多。当今的方法论依赖于将蛋白质混合物分离为其各个成分，然后分析分离的蛋白质。所提出的发展将使混合物中的所有蛋白质同时识别，从而大大减少了分析时间和精力。蛋白质的混合物将被酶消化，并通过液相色谱分离蛋白水解肽的混合物，并通过高分辨率的质谱法分析。原始混合物中的蛋白质将通过使用所产生的精确质量数据从其蛋白水解片段中鉴定出来。目前，只能通过使用准确的质量测量将一小部分肽分配给蛋白质。大多数提出的努力将指向开发方法，以增加可以分配给其母蛋白的肽的比例。提出了一种称为“质量缺陷标签”的方法，以提高分配的特异性。提出了几种新型试剂用于质量缺陷标记，并将作为项目的一部分合成。这些试剂不仅可用于帮助蛋白质识别，而且可以用于执行定量蛋白质组学。其他实验将探索与使用质量缺陷标签的稳定同位素的内源性标记的使用，以实现蛋白质鉴定的高特异性。使用这些方法，计算表明，对于核蛋白蛋白质组的分析，可以将测量的肽的95％分配给其得出的蛋白质。拟议发展的成功将在生物学研究，药物发现和医学中产生重大影响。拟议的努力将由研究生和本科生进行，并将对他们的科学发展有益。参与这项研究的学生将暴露于最先进的高分辨率质谱法。这将为社会提供训练有素的科学家在这个关键技术领域。