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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。系统性初级（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。大多数先前分析的LCs被发现是翻译后修饰的(1)。最常见的情况是除了免疫球蛋白lc中通常发现的分子内二硫键外，c端半胱氨酸的s -半胱氨酸化。我们之前的研究表明，免疫球蛋白LCs的可变区氨基酸替换和一些翻译后修饰（PTMs）可能是通过破坏这些蛋白的折叠状态来促进原纤维形成的关键因素。为了更好地了解LC修饰对淀粉样蛋白沉积的作用，我们使用基于质谱（MS）的方法研究了从诊断为AL淀粉样变性患者的尿液中分离的淀粉样蛋白LC（例如，本文讨论的00-131和00-051），以确定蛋白质中的氨基酸序列变化和PTMs。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；LCs的一些氨基酸替代物和PTMs可能在破坏这些蛋白折叠状态的稳定中起重要作用，从而使它们成为淀粉样蛋白。原子力显微镜用于观察原纤维。在二硫苏糖醇（DTT）处理前后，用纳米喷雾ESI-MS测定完整蛋白的分子质量。样品分液用胰蛋白酶、Asp-N、Lys-C和Glu-C蛋白水解消化。这些酶消化产物的等分被DTT减少。MALDI-MS和ESI-MS分析还原和非还原的消化进行生成肽图。在四极正交TOF质谱上使用ESI-MS/MS或MALDI MS/MS对一些无法通过肽图定位的肽进行测序，这些串联质谱实验也被用于获取ptm的进一步信息。结果与讨论采用透析和凝胶过滤层析纯化了1例患者（00-131）的尿免疫球蛋白LC。其分子量不包含任何与cDNA序列推断的分子量相匹配的成分。还原后检测到分子量为23202和23248的两种组分。在非还原ESI-MS谱中，分子量为23248的组分与cDNA序列相匹配，而分子量为23202的组分比主组分少80 Da。高效液相色谱-质谱分析表明，c端半胱氨酸主要是s -磺化，这解释了还原后观察到的-80 Da位移。这是仅有的第二个s -磺化人类蛋白质的例子。我们以前报道过第一例甲状腺转甲状腺素(2)。还原前，LC 00-051的ESI质谱在24182 Da处有一个主峰。DTT处理后，观察到另一个主峰位于24154 Da，这与从dna推导的氨基酸序列计算出的24156 Da的理论质量很好地一致。此外，还原样品中还存在两种低丰度、低质量的组分。经MS肽图谱和MS/MS测序验证，获得全序列覆盖。鉴定了两个二硫键（Cys23-Cys94, Cys140-Cys200），并阐明了低丰度的s -磺化，以及对N端和c端的新修饰。我们的AFM结果表明，对于样品00-131，蛋白质随时间聚集。在蛋白质聚集过程中也有明显的pH效应，尽管在使用的条件下没有观察到纤维的形成。这些结果与其他测序的淀粉样蛋白形成免疫球蛋白LCs的结果进行了比较，这些LCs表现出翻译后修饰，如同型二聚化、s -半胱氨酸化、n端修饰和糖基化。1. Lim A, Wally J, Walsh MT, Skinner M, Costello CE。分析的。物化学。中文信息学报，2001,29(5):45-56。2. the¿berge R, Skinner M, Connors LH, Skare J., Costello CE, Anal。化学，1999,71:452-459。

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 posttranslationally 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 (e.g., 00-131 and 00-051, discussed herein) 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 an important role in destabilizing the folding state of these proteins, thus making them amyloidogenic. AFM is used to observe fibrils. The molecular masses of the intact protein are determined by 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. These tandem MS experiments are also employed to acquire further information on PTMs. Results and Discussion The urinary immunoglobulin LC from a patient (00-131) was purified using dialysis and gel filtration chromatography. Its molecular weight did not include any components that matched the MW deduced from the cDNA sequence. After reduction, two components of molecular weights 23202 and 23248 were detected. The component of molecular weight 23248 matches that deduced from the cDNA sequence whilst 23202 is 80 Da less than the main component in the non reduced ESI-MS spectrum. HPLC MSMS analysis of the Glu-C digest of the protein indicates that the C-terminal cysteine is primarily S-sulfonated, explaining the -80 Da shift observed after reduction. This is the only the second example of S-sulfonated human protein. We have previously reported the first case in transthyretin (2). Before reduction, the deconvoluted ESI mass spectrum of LC 00-051 showed one major peak at 24,182 Da. After treatment with DTT, another major peak at 24,154 Da was observed, which was in good agreement with the theoretical mass of 24,156 Da calculated from the cDNA-deduced amino acid sequence. In addition, two low-abundance, lower-mass components were present in the reduced sample. The cDNA-deduced sequence was verified by MS peptide mapping and MS/MS sequencing with full sequence coverage obtained. Two disulfide bonds (Cys23-Cys94, Cys140-Cys200) were identified and low abundance S-sulfonation, as well as novel modifications to both the N- and C-termini were elucidated. Our AFM results show that for sample 00-131, the protein aggregates with time. There are also clear pH effects during the protein aggregation process although no fibril formation was observed under the conditions used. These results are being compared with those obtained for other sequenced amyloid-forming immunoglobulin LCs exhibiting posttranslational modifications such as homodimerization, S-cysteinylation, N-terminal modifications and glycosylation. 1. Lim A, Wally J, Walsh MT, Skinner M, Costello CE. Anal. Biochem., 2001, 295, 45-56. 2. Th¿berge R, Skinner M, Connors LH, Skare J., Costello CE, Anal.Chem, 1999, 71 452-459.