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.

项目概要 这里提出的是研究开发一系列技术，使阅读完整的内容成为可能。 飞摩尔色谱时间尺度上蛋白质的氨基酸序列（向前和向后） 水平并表征同一蛋白质分子上存在的多个翻译后修饰 并共同调节其生物活性。这项研究是由我实验室的四项主要创新推动的。这些 包括开发 (a) 用于肽和完整蛋白质断裂的电子转移解离 (ETD)， (b) 结合 ETD 和 IIPT（离子-离子质子转移）化学以获得 n 端和 c 端序列信息 识别蛋白质，(c) 前端 ETD (FETD)，有助于将完整蛋白质的灵敏度提高 10-50 倍 (d) 微柱酶反应器，在 8 M 尿素中完全活跃，可用于生成 来自大蛋白质的 3-10 KDa 蛋白质片段。下一步，我们将实施并优化ETD和IIPT 与平行离子停放 (PIP) 相结合。这将包括引入新的 ETD 试剂和新的 波形可提高平行离子停放的效率，新的 IIPT 试剂可扩展可用质量 范围至 m/z 4,000。还提出了将强大的微柱酶反应器整合到 旋转驱动的微流体盘系统，其中蛋白酶床长度、溶剂流量为数百纳升/分钟，以及 数百毫秒数量级的消化时间都可以高精度地控制和测量。 通过使用旋转驱动的微流控盘系统，我们期望提高样品通量，降低样品 数量，并提高单个样品分析的再现性。所有这一切都将完全应用到 表征（a）双特异性抗体，（b）抗生素耐药性分泌的外膜囊泡中的蛋白质， 革兰氏阴性菌、淋病奈瑟菌、(c) 参与生长或生长抑制的植物蛋白 和 分别是 O-GlcNAcylation 和 O-fucosylation 的靶标，(d) 翻译后修饰 在细胞周期期间存在于组蛋白伴侣上，并且（e）调节的翻译后修饰 组蛋白 H3 和 H4 上的 Arg 残基甲基化。