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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心赠款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 蛋白水解加工是血浆和血清中的关键翻译后修饰。 大多数分泌的蛋白质被蛋白酶处理至少一次，当信号肽被去除时，并且在许多情况下发生额外的处理，例如激素原活化。 此外，释放到血液中的细胞内蛋白质可以被蛋白酶加工，并且这种加工可以指示体内细胞的生理状态;例如，血液中的半胱天冬酶切割的蛋白质可以用作细胞凋亡的标志物。 我们已经开发了一种方法来分析人血清或血浆中的蛋白水解加工，该方法基于使用称为枯草杆菌酶的工程酶对N-末端的特异性标记和富集。 富集后，可以通过LCMSMS鉴定来自蛋白水解加工的蛋白质的N-末端肽。 这种鉴定完全依赖于灵敏的高分辨率质谱仪，包括UCSF质谱设施中的Qstar Elite和Orbitrap仪器。 我们已经在全血清和/或血浆中的约300种蛋白质中鉴定了近1000个独特的N-末端。所鉴定的蛋白质跨越血浆丰度的九个数量级，并且包括补体和凝血因子中的加工位点、注释的信号序列以及激素和生长因子（包括胃抑制肽和血管内皮生长因子）中的加工位点。 特异性蛋白水解裂解肽是良好的生物标志物候选物，我们目前正在进行一项试点研究，以发现接受化疗的弥漫性大B细胞淋巴瘤（DLBCL）患者的治疗效果的肽生物标志物。

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).