PROTEIN CYSTEINE POST-TRANSLATIONAL MODIFICATION IN AMYLOIDOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 半胱氨酸的翻译后修饰可能与某些蛋白质的淀粉样变性有关。我们正在研究这类PTM与两种蛋白质的相关性，这两种蛋白质对淀粉样病非常感兴趣，特别是在BUSM淀粉样治疗和研究中心看到的患者群体的核心，即血清蛋白转甲状腺素和过表达的免疫球蛋白轻链。转甲状腺素(TTR)是一种13.7 kDa的转运蛋白，主要由肝脏合成。在血浆中，四聚体TTR结合视黄醇结合蛋白和甲状腺激素。假设TTR中的氨基酸替代会破坏四聚体的稳定，并导致TTR形成一种中间体，该中间体自身结合成淀粉样纤维。家族性甲状腺激素性淀粉样变性(ATTR)是一种以淀粉样纤维形式存在于各种组织和器官中的遗传性遗传病。目前已鉴定出90多种TTR变异体，其中大多数是淀粉样变性的。由于唯一有效的治疗方法是肝移植，因此正确的临床诊断至关重要。MS资源与淀粉样蛋白治疗和研究计划合作，已经确定了一些具有临床意义的TTR变体的特征。我们现在正在探索QOTOF MS/MS和LTQ-Orbitrap MS/MS的使用，重点是在线信息依赖获取(IDA)毛细管LC MS/MS，通过自动数据库搜索来表征TTR。TTR是从患者血清中免疫沉淀并通过离心法和反相高效液相色谱法纯化而成。进行蛋白质消化，并首先用MALDI-TOFMS进行分析，MALDI-和CapLC-ESI-QOTOFMS/MS在SCHEX/AB QSTAR QOTOF质谱仪上进行。在QStar和LTQ-Orbitrap系统上进行了信息依赖采集(IDA)MS/MS在线毛细管LC-MS。将来自IDA-LC-MS/MS的数据对照Mascot(矩阵科学)和用户编程PRO-ID(ABI)数据库进行分析。质谱仪(MS)在临床诊断中发挥了重要作用。由于人工收集和解释ESI/MALDI/MS/MS数据费时且效率低下，使用蛋白质组学开发的MS/MS方法，如具有自动数据库搜索的IDA-LC-MS/MS，为质谱学的临床应用提供了优势。在突变已被预先编程到数据库中的情况下，获得确凿的变异识别。并且可以使用预先编程的数据库来处理PTM的自动分配，例如半胱氨酸的修饰。同样的方法正在被开发用于半胱氨酸修饰的免疫球蛋白轻链。

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. Post-translational modifications at cysteine may be involved in making certain proteins amyloidogenic. We are investigating the correlation of such PTMs on two proteins that have great interest for the amyloid diseased that are especially central to the patient population seen at the BUSM Amyloid Treatment and Research Center, the serum protein transthyretin and the overexpressed immunoglobulin light chains. Transthyretin (TTR) is a 13.7-kDa transport protein which is synthesized predominantly by the liver. In plasma, tetrameric TTR binds retinol-binding protein and thyroxine. Amino acid substitution in TTR is hypothesized to destabilize the tetramer and cause the TTR to form an intermediate that self associates into amyloid fibrils. Familial transthyretin amyloidosis (ATTR), is associated with the deposition of the TTR variants as amyloid fibrils in various tissues and organs. More than 90 TTR variants have been identified, with the majority being amyloidogenic. Since the only effective treatment of ATTR is liver transplantation, the correct clinical diagnosis is critical. The MS Resource, in collaboration with the Amyloid Treatment and Research Program, has characterized a number of TTR variants of clinical significance. We are now exploring the use of QoTOF MS/MS and LTQ-Orbitrap MS/MS with emphasis on on-line information dependent acquisition (IDA) capillary LC MS/MS for the characterization of TTR via automated data-base searching. TTR is immunoprecipitated from the serum of patients and purified by centrifugation and reversed phase HPLC. Proteolytic digestions are performed and the digests are first analyzed by MALDI-TOFMS MALDI- and capLC-ESI-QoTOFMS/MS are performed on an Sciex/AB QSTAR QoTOF mass spectrometer. On-line capillary LC-MS with information dependent acquisition (IDA) MS/MS is performed on the QStar and LTQ-Orbitrap systema. Data from IDA-LC-MS/MS are analyzed against Mascot (Matrix Science) and user programmed PRO-ID (ABI) databases. Mass spectrometry (MS) has played an important role in the clinical diagnosis of ATTR. Since manual collection and interpretation of ESI/MALDI/MS/MS data is time consuming and inefficient, use of proteomics developed MS/MS methods, such as IDA-LC-MS/MS with automated database searching, offers advantages to the clinical applications of mass spectrometry. Conclusive variant identification is obtained in cases where the mutation has been pre-programmed into the database.and automated assignment of PTMs such as modifications at cysteine can be handled using the preprogrammed database. The same approach is being developed for cysteine-modified IgG light chains.