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

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。所列机构为中心，不一定是研究者所在机构。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。大多数先前分析的LC已被发现是后修饰的（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）的方法来研究淀粉样蛋白生成LC（例如，00-131和00-051，本文讨论），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用于观察原纤维。 在用二硫苏糖醇（DTT）处理之前和之后，通过纳米喷雾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。一些不能通过肽图谱分配的肽使用ESI-MS/MS或MALDI MS/MS在四极杆正交TOF MS上进行测序。这些串联MS实验也用于获得关于PTM的进一步信息。 结果和讨论使用透析和凝胶过滤色谱法纯化来自患者（00-131）的尿免疫球蛋白LC。其分子量不包括与从cDNA序列推导的MW相匹配的任何组分。还原后，检测到分子量为23202和23248的两种组分。分子量23248的组分与从cDNA序列推断的组分匹配，而23202比非还原ESI-MS光谱中的主要组分小80 Da。蛋白质的Glu-C消化物的HPLC MSMS分析表明，C-末端半胱氨酸主要是S-磺化的，解释了还原后观察到的~ 80 Da位移。这是唯一的第二个S-磺化人类蛋白质的例子。我们以前曾报道过第一例甲状腺素运载蛋白（2）。 还原前LC 00-051的去卷积ESI质谱在24，182 Da处显示一个主峰，DTT处理后在24，154 Da处观察到另一个主峰，与cDNA推导的氨基酸序列计算的理论质量24，156 Da一致。此外，在还原样品中存在两种低丰度、低质量组分。通过MS肽图和MS/MS测序验证cDNA推导的序列，获得全序列覆盖。鉴定了两个二硫键（Cys 23-Cys 94，Cys 140-Cys 200），并阐明了低丰度的S-磺化，以及对N-和C-末端的新修饰。 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，尽管在所用条件下没有观察到原纤维形成。这些结果正在与其他测序的淀粉样蛋白形成免疫球蛋白LC表现出翻译后修饰，如同源二聚化，S-半胱氨酸化，N-末端修饰和糖基化得到的结果进行比较。 1. Lim A，Wally J，沃尔什MT，Skinner M，Costello CE. Anal.生物化学，2001，295，45-56. 2. Th berge R，Skinner M，Connors LH，斯卡雷J.，Costello CE，Anal.Chem，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.