HOMOCYSTEINYLATED TRANSTHYRETIN IN HUMAN PLASMA BY ELECTROSPRAY IONIZATION MS

通过电喷雾电离 MS 检测人血浆中同型半胱氨酸化转甲状腺素蛋白

• 批准号: 7369213
• 负责人: Donald Weldon Jacobsen
• 金额: $ 0.05万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369213
• 关键词: HOMOCYSTEINYLATED; TRANSTHYRETIN; HUMAN; PLASMA; ELECTROSPRAY

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Elevation in the level of total plasma homocysteine (Hcys) is a strong independent risk factor for cardiovascular disease and an emerging risk factor for Alzheimer¿s disease. Although over 70% of total Hcys is disulfide linked to plasma proteins, little is known about the identity of the carrier proteins the effect(s) of homocysteinylation on protein function. Recently, we reported that the amyloidogenic protein transthyretin (TTR, prealbumin) carries Hcys by disulfide linkage at Cys10. SDS-PAGE analysis of 35S-L-homocysteine radiolabeled TTR purified from human plasma revealed two bands corresponding to dimeric (27.5 kDa) and tetrameric (55 kDa) TTR. Treatment of the radiolabeled sample with b-mercaptoethanol prior to SDS-PAGE analysis removed the radiolabel from TTR, confirming the presence of a disulfide linkage. ESI MS analysis of the TTR immunoprecipitated from human plasma (without DTT treatment) provides unambiguous evidence that TTR is a carrier for Hcys. TTR-Cys10-S-S-Hcys (homocysteinylated) is readily detectable in plasma from healthy individuals and patients suffering from end-stage renal disease and homocystinuria. In vitro studies on dose-dependent homocysteinylation of TTR showed that the level of homocysteinylated TTR in plasma is directly proportional to the amount of L-Hcys (0-500 ¿M) added. A similar correlation was found between total Hcys concentration and homocysteinylated TTR in normal and hyperhomocysteinemic plasma. Thus, homocysteinylated TTR appears to be a sensitive indicator of Hcys burden. The results were published in J. Biol. Chem. during the last reporting year. The patent application for a diagnostic test based on this work is slowly progressing through the system.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。血浆总高半胱氨酸（Hcys）水平升高是心血管疾病的一个强独立危险因素，也是阿尔茨海默病的一个新出现的危险因素。虽然超过70%的总半胱氨酸是二硫键连接的血浆蛋白，很少知道的身份载体蛋白的同型半胱氨酸化对蛋白质功能的影响。最近，我们报道了淀粉样蛋白转甲状腺素蛋白（TTR，前白蛋白）携带的半胱氨酸的二硫键在Cys 10。从人血浆中纯化的35 S-L-高半胱氨酸放射性标记TTR的SDS-PAGE分析显示了对应于二聚体（27.5 kDa）和四聚体（55 kDa）TTR的两条带。在SDS-PAGE分析前，用b-巯基乙醇处理放射性标记的样品，去除TTR中的放射性标记，证实存在二硫键。对从人血浆（未经DTT处理）中免疫沉淀的TTR的ESI MS分析提供了明确的证据，证明TTR是Hcys的载体。TTR-Cys 10-S-S-Hcys（同型半胱氨酸化）在健康个体和患有终末期肾病和同型半胱氨酸尿症的患者的血浆中容易检测到。TTR剂量依赖性同型半胱氨酸化的体外研究表明，血浆中同型半胱氨酸化TTR的水平与加入的L-Hcys（0-500 μ M）的量成正比。在正常和高同型半胱氨酸血症血浆中，总同型半胱氨酸浓度和同型半胱氨酸化TTR之间存在类似的相关性。因此，同型半胱氨酸化TTR似乎是HCys负荷的敏感指标。结果发表在上一报告年度的《生物化学杂志》上。基于这项工作的诊断测试的专利申请正在缓慢地通过系统进行。