PROTEOMIC IDENTIFICATION OF PROTEIN N-TERMINI IN BLOOD PLASMA AND SERUM

血浆和血清中蛋白质 N 末端的蛋白质组学鉴定

• 批准号: 8363786
• 负责人: JAMES A WELLS
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363786
• 关键词: Apoptosis; Biological Markers; Blood; Blood Coagulation Factor; Caspase; Cells; Cleaved cell; Complement; Engineering; Enzymes; Funding; Gastric Inhibitory Polypeptide; Grant; Growth Factor; Hormones; Human; Label; Mass Spectrum Analysis; Methods; N-terminal; National Center for Research Resources; Patients; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Physiological; Pilot Projects; Plasma; Post-Translational Protein Processing; Principal Investigator; Process; Proteins; Proteolytic Processing; Proteomics; Research; Research Infrastructure; Resolution; Resources; Serum; Site; Source; Treatment Efficacy; United States National Institutes of Health; Vascular Endothelial Growth Factors; base; chemotherapy; cost; instrument; large cell Diffuse non-Hodgkin' s lymphoma; mass spectrometer; prohormone; subtiligase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Proteolytic processing is a key post-translational modification in blood plasma and serum. Most secreted proteins are processed by proteases at least once, when the signal peptide is removed, and additional processing occurs in many cases, such as prohormone activation. Also, intracellular proteins released into the blood may be processed by proteases, and this processing may be indicative of physiological status of cells in the body; for example, caspase-cleaved proteins in the blood may serve as markers of apoptosis. We have developed a method to profile proteolytic processing in human serum or plasma, based on specific labeling and enrichment of N-termini using an engineered enzyme called subtiligase. After enrichment, N-terminal peptides from proteolytically processed proteins can be identified by LCMSMS. This identification is absolutely depended on sensitive and high-resolution mass spectrometers including the Qstar Elite and Orbitrap instruments in the UCSF Mass Spectrometry Facility. We have identified nearly 1000 unique N-termini in about 300 proteins in whole serum and/or plasma. The identified proteins span nine orders of magnitude in plasma abundance, and include processing sites in complement and coagulation factors, annotated signal sequences, and processing sites in hormones and growth factors, including Gastric Inhibitory Peptide and Vascular Endothelial Growth Factor. Specific proteolytically cleaved peptides are good biomarker candidates and we are currently conducting a pilot study to discover peptide biomarkers of treatment efficacy in patients undergoing chemotherapy for diffuse large B-cell lymphoma (DLBCL).

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 蛋白水解物是血浆和血清中一种关键的翻译后修饰。当信号肽被移除时，大多数分泌的蛋白质至少被蛋白酶处理一次，在许多情况下，还会发生额外的处理，如激素原激活。此外，释放到血液中的细胞内蛋白可能会被蛋白酶处理，这种处理可能表明体内细胞的生理状态；例如，血液中被caspase裂解的蛋白可能是细胞凋亡的标志。我们已经开发了一种方法来描述人血清或血浆中的蛋白降解过程，该方法基于一种名为枯草杆菌酶的工程酶对N-末端的特定标记和浓缩。经过浓缩后，可以用LCMMS鉴定蛋白质的N-末端多肽。这种鉴定完全依赖于灵敏和高分辨率的质谱仪，包括加州大学旧金山分校质谱分析设施中的QStar Elite和Orbitrap仪器。我们已经在全血清和/或血浆中的大约300个蛋白质中确定了近1000个唯一的N末端。已鉴定的蛋白质跨越血浆丰度的九个数量级，包括补体和凝血因子的加工位点，注释的信号序列，以及激素和生长因子的加工位点，包括胃抑制肽和血管内皮生长因子。特定的蛋白水解肽是很好的生物标记物候选者，我们目前正在进行一项试点研究，以发现对正在接受化疗的弥漫性大B细胞淋巴瘤(DLBCL)患者的疗效的多肽生物标志物。