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 资助的中心拨款提供的资源的众多研究子项目之一，该子项目和研究者 (PI) 可能已从其他 NIH 来源获得主要资助，因此可以在其他 CRISP 机构中得到体现。列出的是该中心，不一定是研究者的机构。血浆总同型半胱氨酸 (Hcys) 水平的升高是心血管疾病的一个强大的独立危险因素，也是阿尔茨海默病的一个新兴危险因素。尽管超过 70% 的总 Hcy 是与血浆蛋白相连的二硫键，但我们对载体蛋白的身份以及同型半胱氨酸化对蛋白功能的影响知之甚少。前白蛋白）通过 Cys10 上的二硫键携带 Hcys，对从人血浆中纯化的 35S-L-同型半胱氨酸进行 SDS-PAGE 分析。显示了对应于二聚体 (27.5 kDa) 和四聚体 (55 kDa) TTR 的两条条带。在 SDS-PAGE 分析之前用 b-巯基乙醇处理放射性标记样品，从 TTR 中去除放射性标记，证实了 ESI MS 分析的存在。从人血浆中免疫沉淀的 TTR（未经 DTT 处理）提供了明确的证据，证明 TTR 是 Hcys 的载体。 TTR-Cys10-S-S-Hcys（同型半胱氨酸）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很容易检测到。 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的 TTR 水平为与 L-Hcys 的量成正比 (0-500 ¿ M）补充说，在正常和高同型半胱氨酸血浆中，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。基于这项工作的诊断测试的专利申请正在系统中缓慢进展。