Sequential ion/ion reactions for large peptide and whole protein characterization

用于大肽和整个蛋白质表征的连续离子/离子反应

• 批准号: 7585767
• 负责人: JOSHUA J COON
• 金额: $ 24.91万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7585767
• 关键词: American; Anions; Biological; Charge; Chimeric Proteins; Chromatography; Classification; Codon Nucleotides; Collection; Coupled; Coupling; Data; Development; Digestion; Dissociation; Electron Transport; Eukaryota; Evolution; Family; Foundations; Gene Fusion; Generations; Genes; Human; Hybrids; Individual; Influentials; Ions; Length; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Methodology; Methods; Modality; Operative Surgical Procedures; Pathway interactions; Pattern; Peptides; Phase; Post-Translational Protein Processing; Process; Prostate; Proteins; Proteomics; Protons; Proxy; RNA Splicing; Reaction; Recurrence; Research Personnel; Resolution; Screening procedure; Sequence Analysis; Single Nucleotide Polymorphism; System; TMPRSS2 gene; Techniques; Technology; Tertiary Protein Structure; Time; Trypsin; Variant; Work; base; chemical reaction; design; genome sequencing; instrument; instrumentation; male; mass spectrometer; new technology; novel; programs; reaction rate; segregation; success; tandem mass spectrometry; tool

DESCRIPTION (provided by applicant): Perhaps one of the most influential concepts in protein mass spectrometry has been the notion of enzymatic protein digestion to render a collection of peptides of suitable size for conventional tandem mass spectrometry (collisional-activation, CAD).1 Doubtless this methodology has enabled significant progress for global protein identification; however, many investigators now realize this approach has significant limitations.2 This conclusion is based upon the following observations: First, protein posttranslational modifications (PTMs) on multi-domain proteins, and among components of protein-protein machines, work in concert; to determine their biological relevance, these patterns must be detected within the context of one another (across the whole protein).3 Second, transcriptional editing processes are pervasive in higher eukaryotes and difficult to predict, even with a completely sequenced genome. For example, 3/4 of all human proteins are expected to have at least 1 splice variant4-6 - variants that could contain intronic sequences. Skipped codons, frameshifting, gene fusion, and single nucleotide polymorphisms (SNPs) also occur. Thus, the use of short peptides as proxy markers for genes is inadequate and often misleading. Unlike CAD, electron transfer dissociation (ETD), a new fragmentation technique co-invented by the PI, does not require short peptides for successful sequence analysis (i.e., trypsin digestion). ETD is indifferent to peptide length or the presence of PTMs, is performed on a time-scale that permits coupling with chromatography, and can be coupled to other ion/ion reactions. This proposal aims to develop a suite of core ion/ion reaction tools, and automate their use in a hybrid-

描述（由申请人提供）：也许蛋白质质谱法中最有影响力的概念之一是酶促蛋白质消化的概念，以提供用于常规串联质谱法的合适大小的肽集合（碰撞激活，CAD）。1毫无疑问，这种方法使全球蛋白质鉴定取得了重大进展;然而，许多研究人员现在意识到这种方法有很大的局限性。2这一结论是基于以下观察：首先，多结构域蛋白质上的蛋白质翻译后修饰（PTMs）以及蛋白质-蛋白质机器的组成部分之间协同工作;为了确定它们的生物学相关性，这些模式必须在彼此的背景下（在整个蛋白质中）被检测到。3第二，转录编辑过程在高等真核生物中是普遍存在的，并且难以预测，即使有完全测序的基因组。例如，所有人类蛋白质的3/4预期具有至少1个可能含有内含子序列的剪接变体4 -6 -变体。密码子跳跃、移码、基因融合和单核苷酸多态性（SNP）也会发生。因此，使用短肽作为基因的替代标记物是不充分的，并且经常误导。与CAD不同，电子转移解离（ETD），一种由PI共同发明的新片段化技术，不需要短肽进行成功的序列分析（即，胰蛋白酶消化）。ETD与肽长度或PTM的存在无关，在允许与色谱偶联的时间尺度上进行，并且可以与其他离子/离子反应偶联。该提案旨在开发一套核心离子/离子反应工具，并在混合动力系统中自动使用它们，