Bio-Organic Biomedical Mass Spectromy Resource

生物有机生物医学质谱资源

基本信息

批准号：
8416624
负责人：
ALMA L BURLINGAME
金额：
$ 43.25万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-03-15 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8416624
关键词：
Address Affinity Algorithms Amination Amines Architecture Binding Biochemical Bioinformatics Biomedical Research Cations Cells Characteristics Charge Chemicals Chromatography Communities Complex Computer software Cross-Linking Reagents DNA Polymerase II Data Set Databases Development Diagnostic Digestion Dissociation Electron Transport Electrospray Ionization Frequencies Gases Genetic Transcription HIV Health Homeostasis Homology Modeling Human Human Biology Hydrazine Imagery Infectious Agent Ions Laboratories Length Lysine Malignant Neoplasms Maps Mass Spectrum Analysis Methodology Methods Modeling Modification Molecular Nature Peptide Hydrolases Peptides Pharmaceutical Preparations Phase Plant Resins Principal Investigator Process Proteins Proteomics Protocols documentation Public Health RNA Polymerase II Randomized Reaction Reagent Recovery Reporting Research Resolution Resources Side Signal Transduction Site Solubility Speed Stem cells Streptavidin Structure Techniques Technology Tissues Toxin Transcription Initiation Trypsin United States National Institutes of Health Universities Vertebral column arm base cost crosslink molecular size polypeptide protein complex stoichiometry tandem mass spectrometry tool

项目摘要

DESCRIPTION (provided by applicant): This revision of NIH P41RR001614 requests support to expand our development of a new class of chemical cross-linking reagents to address new Biodriver needs for integrated, robust methodology that will yield distance constraints for modeling of the architecture of much larger protein complexes and machines. The new Biodriver Project is entitled, "Architecture of RNA polymerase II Transcription Initiation Complexes," the Principal Investigator is Roger Kornberg of Stanford University. Our plan is to build upon our recent successful TR&D demonstration that reductive amination of aryldialdehydes with e-aminofunctions on lysine side chains is virtually ideally suited to sequence and structural characterization by electron transfer dissociation (ETD) mass spectrometry using the Velos Orbitrap platform. Virtually complete sequences are obtained from ETD spectra for BOTH peptides in the cross-linked species. Also gas phase enrichment of peptide cross-links Is achieved by selection of precursor charge state (eliminate states below +4) for ETD spectral recording, this is possible because reductive amination generates two new secondary amines that are protonated in electrospray ionization. Kornberg's laboratory has highly purified complexes that range in molecular size from some 30 proteins to more than 100. Taking advantage of this challenging array of related complexes, we will be able to optimize and exploit the major inherent advantages of our new integrated experimental strategy (reagents, optimal conditions for cross-linking and subsequent digestion, ETD MS and new software and bioinformatics tools to facilitate and accelerate progress). Once a robust protocol is optimized using our new integrated strategy, this capability will be exported to the biomedical research community. Almost untold numbers of such protein complexes exist that carry out a host of processes and functions in human and other cells. For example, we have just reported the characterization of the HIV-human host protein interactome landscape that provides evidence of almost 500 complexes (Jaeger et al., 2012 Nature, 481, 365-370,doi:10.1038/nature10719).

描述（由申请人提供）：NIH P41RR001614的此修订要求支持我们开发我们对新的化学交联试剂的开发，以满足集成，可靠的方法的新生物层需求，该方法将产生距离距离的距离约束，以建模更大的蛋白质复合物和机器的建筑。新的生物流程项目的标题为“ RNA聚合酶II转录启动综合体的建筑”，主要研究人员是斯坦福大学的Roger Kornberg。我们的计划是基于我们最近成功的TR＆D证明，即用丝氨酸侧链上的E-氨基醛还原性氨基化的胺化，实际上非常适合通过使用Velos OrbitRap平台通过电子传递分离（ETD）质谱法来序列和结构表征。几乎完整的序列是从交联物种中的两个肽的ETD光谱中获得的。同样，通过选择ETD光谱记录的前体电荷态（消除+4以下的状态），可以实现肽交联的气相富集，这是可能的，因为还原性胺化会产生两个在电喷雾电离中质子化的新次级胺。 Kornberg的实验室具有高度纯化的复合物，其分子大小从约30种蛋白质到100多个。利用这些具有挑战性的相关复合物，我们将能够优化和利用我们新的集成实验策略的主要固有优势（重新恢复，重新链接，最佳的交叉链接条件，用于交叉链接和随后的eTDSTECTIC和NEW SOFTART和NEW SOVETICS和BIOEDER和BIOEDERICT和BIOETERITISIC。一旦使用我们的新集成策略优化了强大的协议，该功能将出口到生物医学研究界。存在几乎数量的此类蛋白质复合物，这些复合物在人和其他细胞中进行了许多过程和功能。例如，我们刚刚报道了HIV-Human宿主蛋白质相互作用的景观的表征，该景观提供了近500个复合物的证据（Jaeger等，2012 Nature，481，365-370，doi：10.1038/nature10719）。