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

诊断为原发性淀粉样变性患者的淀粉样变性轻链的 MS CHAR

• 批准号: 7722977
• 负责人: DAVID C SELDIN
• 金额: $ 2.85万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722977
• 关键词: Aftercare; Aliquot; Amino Acid Sequence; Amino Acids; Amyloid Fibrils; Amyloid deposition; Amyloidosis; B-Lymphocytes; C-terminal; Computer Retrieval of Information on Scientific Projects Database; Condition; Cysteine; Diagnosis; Digestion; Dithiothreitol; Funding; Grant; Homodimerization; Immunoglobulin Light Chain Deposition; Immunoglobulin Variable Region; Immunoglobulins; In Vitro; Institution; Light; Light-Chain Immunoglobulins; Mass Spectrum Analysis; Methods; Modification; N-terminal; Patients; Peptide Mapping; Peptides; Play; Post-Translational Protein Processing; Process; Proteins; Research; Research Personnel; Resources; Role; Sampling; Source; Spectrometry; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time; Tissues; Trypsin; United States National Institutes of Health; Urine; Variant; base; disulfide bond; glycosylation; immunoglobulin deposition disease; molecular mass; primary amyloidosis of light chain type; protein aggregate; protein aggregation; research study; tandem mass spectrometry

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 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. 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.

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。 全身性原发(AL，免疫球蛋白轻链)淀粉样变性的特征是由单克隆性B细胞克隆产生的免疫球蛋白轻链(LC)蛋白沉积。之前分析的大多数翻译后修饰词汇被发现(1)。最常见的情况是除了免疫球蛋白LCS中通常存在的分子内二硫键外，C端半胱氨酸的S半胱氨酸化。我们以前的研究表明，免疫球蛋白LCS可变区的氨基酸替换和一些翻译后修饰(PTM)可能是通过破坏这些蛋白质的折叠状态而导致纤维形成的关键因素。为了更好地了解LC修饰在淀粉样蛋白沉积中的作用，我们使用基于质谱仪(MS)的方法来研究从确诊为AL淀粉样变性的患者尿液中分离出的致淀粉样LC，以确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控制其形成淀粉样纤维的关键，LCS上的一些氨基酸替代和PTM可能在破坏这些蛋白质的折叠状态从而使其形成淀粉样蛋白方面发挥重要作用。原子力显微镜被用来观察在不同条件下从患者组织和其他体外生长的组织中获得的纤维。 用纳米级ESI-MS测定二硫苏糖醇(DTT)处理前后完整蛋白的相对分子质量。样品等分用胰酶、天冬氨酸-N、赖氨酸-C和谷氨酸-C消化。这些酶消化产物的等量用DTT还原。对还原和非还原的消化进行MALDI-MS和ESI-MS分析，以生成肽图。利用ESI-MS/MS或MALDI-MS/MS在四极正交飞行时间质谱仪上对一些不能通过肽图谱进行指认的多肽进行了测序，这些串联的MS实验也被用来获得关于PTMS的进一步信息。 AFM结果表明，LC蛋白随着时间的延长而聚集。在蛋白质聚集过程中也存在明显的pH效应。这些来自不同患者样本的结果正在进行比较；这些样本包含许多翻译后修饰，如同源二聚、S-半胱氨酸化、N-末端修饰和糖基化。