MS CHAR OF AMYLOIDOGENIC LIGHT CHAINS OF PTS DIAGNOSED W/PRIMARY AMYLOIDOSIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 系统性原发性（AL，免疫球蛋白轻链）淀粉样变性的特征是由单克隆B细胞衍生克隆产生的免疫球蛋白轻链（LC）蛋白沉积。已经发现大多数先前分析的LC是后修饰的（1）。最常见的情况是除了通常在免疫球蛋白LC中发现的分子内二硫键之外，C-末端半胱氨酸的S-半胱氨酸化。我们以前的研究表明，可变区的氨基酸替换和一些翻译后修饰（PTM）的免疫球蛋白LC可能是通过破坏这些蛋白质的折叠状态，有助于纤维形成的关键因素。为了更好地理解LC修饰对淀粉样蛋白沉积的作用，我们使用基于质谱（MS）的方法来研究从诊断为AL淀粉样变性的患者的尿液中分离的淀粉样LC，以鉴定蛋白质中的氨基酸序列变异和PTM。免疫球蛋白LC的折叠稳定性通常被认为是其形成淀粉样原纤维倾向的控制关键; LC的一些氨基酸替换和PTM可能在使这些蛋白质的折叠状态不稳定中起重要作用，从而使它们成为淀粉样蛋白原。 AFM用于观察从患者组织和在各种条件下体外生长的其他组织中获得的原纤维。 在用二硫苏糖醇（DTT）处理之前和之后，通过MALDI-MS和/或纳米喷雾ESI-MS测定完整蛋白质的分子量。用胰蛋白酶、Asp-N、Lys-C和Glu-C对样品等分试样进行蛋白水解消化。用DTT还原这些酶消化产物的等分试样。对还原和非还原肽进行MALDI-MS和ESI-MS分析，以生成肽图谱。使用ESI-MS/MS或MALDI MS/MS在四极杆正交TOF MS和/或LTQ-Orbitrap MS系统上对一些无法通过肽图谱分配的肽进行测序。这些串联MS实验也被用来获得更多的信息PTM。 我们的AFM结果表明，LC蛋白聚集与时间。在蛋白质聚集过程中也存在明显的pH效应。正在比较来自不同患者样本的这些结果;样本含有许多翻译后修饰，如同源二聚化、S-半胱氨酸化、N-末端修饰和糖基化。描述最近结果的手稿正在由J. Biol. Chem.进行审查。与糖胺聚糖相互作用的特异性也正在研究中。

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. Systemic Primary (AL, immunoglobulin light chain) amyloidosis is characterized by the deposition of immunoglobulin light chain (LC) proteins produced by a monoclonal B-cell-derived clone. Most of the previously analyzed LCs have been found to be post-translationally modified (1). The most common case is S-cysteinylation of the C-terminal cysteine in addition to the intramolecular disulfide bonds normally found in immunoglobulin LCs. Our previous studies have shown that the amino acid replacements in the variable region and some post-translational modifications (PTMs) of immunoglobulin LCs may be the key factors that contribute to fibril formation by destabilizing the folding state of these proteins. To have a better understanding of the role of LC modifications on amyloid deposition, we use a mass spectrometry (MS) based method to investigate amyloidogenic LCs isolated from the urine of patients diagnosed with AL amyloidosis, to identify amino acid sequence variations and PTMs in the protein. The folding stability of an immunoglobulin LC is generally regarded as a controlling key of its tendency to form amyloid fibrils; some amino acid replacements and PTMs of LCs may play important roles in destabilizing the folding state of these proteins, thus making them amyloidogenic. AFM is used to observe fibrils obtained from patient tissues and others grown in vitro under various conditions. The molecular masses of the intact protein are determined by MALDI-MS and/or nanospray ESI-MS, before and after treatment with dithiothreitol (DTT). The sample aliquots are proteolytically digested with trypsin, Asp-N, Lys-C, and Glu-C. Aliquots of these enzymatic digestion products are reduced with DTT. MALDI-MS and ESI-MS analyses of both reduced and non-reduced digests are performed to generate peptide maps. Some peptides that cannot be assigned by peptide mapping are sequenced using ESI-MS/MS or MALDI MS/MS on a quadrupole orthogonal TOF MS and/or the LTQ-Orbitrap MS system. These tandem MS experiments are also employed to acquire further information on PTMs. Our AFM results show that the LC protein aggregates with time. There are also clear pH effects during the protein aggregation process. These results from different patient samples are being compared; the samples contain many post-translational modifications such as homodimerization, S-cysteinylation, N-terminal modifications and glycosylation. A manuscript describing recent results is under review by J. Biol. Chem. The specificity of interactions with glycosaminoglycans is being investigated as well.