Protein Sequencing by Tandem Mass Spectrometry

通过串联质谱法进行蛋白质测序

• 批准号: 10436818
• 负责人: DONALD F HUNT
• 金额: $ 75.71万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-12 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10436818
• 关键词: Acetylation; Affect; Amino Acid Sequence; Animals; Antibiotic Resistance; Arginine; Bacterial Proteins; Beds; Biological; Bispecific Antibodies; C-terminal; Cell Cycle; Cell physiology; Charge; Chemistry; Chromatin; Chromatography; Collaborations; Collection; Dangerousness; Development; Diagnostic; Digestion; Dissociation; Electron Transport; Family; Frequencies; Fucosyltransferase; Funding; Generations; Genes; Genetic Transcription; Genome; Goals; Gram-Negative Bacteria; Growth; High Frequency Oscillation; Histone H3; Histone H4; Histones; Individual; Ions; Lead; Length; Mass Spectrum Analysis; Measures; Medicine; Membrane; Membrane Proteins; Methods; Methylation; Microbiology; Microfluidics; Molecular Chaperones; N-terminal; Napping; Neisseria gonorrhoeae; O-GlcNAc transferase; Ocular orbit; Organism; Parasites; Parents; Peptide Hydrolases; Peptide Sequence Determination; Peptides; Pharmaceutical Preparations; Phosphorylation; Plant Proteins; Plants; Post-Translational Protein Processing; Protein Fragment; Proteins; Protons; Reagent; Reproducibility; Research; Role; Rotation; Running; Sampling; Solvents; Superbug; System; Technology; The Sun; Time; Toxoplasma gondii; Transcriptional Regulation; United States National Institutes of Health; Universities; Urea; Vesicle; Work; biological systems; cell growth; college; enzyme reactor; innovation; instrumentation; millisecond; new technology; novel therapeutics; plant growth/development; professor; tandem mass spectrometry; vaccine development

Project Summary Proposed here is research to develop a collection of technologies that will make it possible to read the complete amino acid sequence of proteins (forwards and backwards) on a chromatographic time scale at the femtomole level and to also characterize multiple posttranslational modifications that exist on the same protein molecule and together regulate its biological activity. This research is driven by four major innovations from my lab. These include development of (a) electron transfer dissociation (ETD) for fragmentation of peptides and intact proteins, (b) combined ETD and IIPT (ion-ion proton transfer) chemistry to obtain n- and c-terminal sequence information that identifies proteins, (c) front end ETD (FETD) that facilitates a 10-50 fold increase in sensitivity for intact proteins, and (d) a micro column enzyme reactor that is fully active in 8 M urea and can be employed to generate 3-10 KDa protein fragments from large proteins. Going forward, we will implement and optimize ETD and IIPT in combination with parallel ion parking (PIP). This will include the introduction of new ETD reagents and new wave forms to enhance the efficiency of parallel ion parking and new IIPT reagents to extend the usable mass range to m/z 4,000. Also proposed is research to incorporate the powerful micro-column enzyme reactor into a rotation-driven microfluidic disc system where the protease bed length, solvent flow in hundreds of nL/min, and digestion times on the order of hundreds of milliseconds can all be controlled and measured with high accuracy. By using the rotation-driven microfluidic disc system, we expect to increase sample throughput, lower sample quantities, and increase reproducibility of individual sample analyses. All of this will be applied to fully characterize (a) bispecific antibodies, (b) proteins in outer membrane vesicles secreted by the antibiotic resistant, gram negative bacteria, Neisseria gonorrhoeae, (c) plant proteins that are involved in growth or growth inhibition and are targets for O-GlcNAcylation and O-fucosylation, respectively, (d) posttranslational modifications that exist on histone chaperones during the cell cycle, and (e) posttranslational modifications that regulate methylation of Arg residues on histones H3 and H4.

项目摘要 这里提出的研究是开发一系列技术的研究，这些技术将使阅读完整 蛋白质的氨基酸序列（正向和向后）在themole的色谱时间尺度上 水平，还表征了在同一蛋白质分子上存在的多种翻译后修饰 并共同调节其生物学活性。这项研究是由我实验室的四项主要创新驱动的。这些 包括开发（a）电子转移解离（ETD），用于肽和完整蛋白的破碎， （b）联合ETD和IIPT（离子质子质子转移）化学以获得N-和C末端序列信息 鉴定蛋白质，（c）前端ETD（FETD），可促进完整的灵敏度增强10-50倍 蛋白质和（d）在8 m尿素中完全活跃的微柱酶反应器，可用于生成 来自大蛋白的3-10 kDa蛋白片段。展望未来，我们将实施并优化ETD和IIPT 结合平行离子停车场（PIP）。这将包括引入新的ETD试剂和新的试剂 波形以提高平行离子停车和新的IIPT试剂的效率以扩展可用的质量 范围为m/z 4,000。还提出的研究是将强大的微甲酶反应器纳入一个 旋转驱动的微流体盘系统，其中蛋白酶床的长度，数百个NL/min的溶剂流量，以及 在数百毫秒顺序上的消化时间都可以高精度控制和测量。 通过使用旋转驱动的微流体盘系统，我们希望增加样品吞吐量，较低的样品 数量并增加单个样本分析的可重复性。所有这些都将应用于完全 表征（a）双特异性抗体，（b）外膜囊泡中抗生素抗生素分泌的蛋白， 革兰氏阴性细菌，淋病奈瑟菌，（c）参与生长或生长抑制的植物蛋白 并且分别是O-Glcnacylation和O-凝集糖基化的靶标，（D）翻译后修饰 在细胞周期中存在于组蛋白伴侣，以及（e）调节后翻译后修饰 H3和H4的ARG残基的甲基化。