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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。总血浆同型半胱氨酸（HCY）的升高是心血管疾病的强大独立危险因素，也是阿尔茨海默氏病的新兴危险因素。尽管超过70％的总HCY是与血浆蛋白相关的二硫化物，但对载体蛋白的身份鲜为nation蛋白对蛋白质功能的影响知之甚少。最近，我们报道说，淀粉样蛋白转甲状腺素蛋白（TTR，PREALBUMIN）通过Cys10的二硫键携带HCY。从人等离子体纯化的35s-L-型摩cass体标记的TTR的SDS-PAGE分析揭示了两个对应于二聚体（27.5 kDa）和四聚体（55 kDa）ttr的条带。在进行SDS-PAGE分析之前，用B-甲醇乙醇处理放射性标记可从TTR中取出放射性标记，从而确认存在二硫键的存在。从人血浆（未经DTT处理）中对TTR免疫沉淀的TTR分析提供了明确的证据，表明TTR是HCYS的载体。在健康个体和患有终末期肾脏疾病和同囊肿的患者的血浆中，很容易检测到TTR-CYS10-S-S-S-HCYS（同型囊藻化）。在TTR的剂量依赖性同叶酸的体外研究表明，等离子体中的同叶酸TTR水平与添加的L-HCYS（0-500€）的量成正比。在正常和多层结晶血浆中，总HCYS浓度与同型固醇的TTR之间发现了类似的相关性。这就是同型凝结的TTR似乎是伯恩的敏感指标。结果发表在J. Biol中。化学在上一个报告年度。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。