PROTEOMIC APPROACH TO THE STUDY OF SYSTEMIC AMYLOIDOSIS

系统性淀粉样变性研究的蛋白质组学方法

• 批准号: 7723067
• 负责人: GIAMPAOLO MERLINI
• 金额: $ 0.97万
• 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/7723067
• 关键词: Abdomen; Affect; Amyloid; Amyloid deposition; Amyloidosis; Appearance; Aspirate substance; Buffers; Categories; Computer Retrieval of Information on Scientific Projects Database; Computer software; Deposition; Diagnosis; Diagnostic; Digestion; Disease; Endopeptidases; Fatty acid glycerol esters; Fingerprint; Functional disorder; Funding; Gel; Grant; Image; Individual; Institution; Location; Maps; Medical; Methodology; Molecular; Organ; Patients; Peptide Hydrolases; Peptides; Personal Satisfaction; Post-Translational Protein Processing; Proteins; Proteomics; Research; Research Personnel; Resources; Sampling; Silver Staining; Source; Spectrum Analysis; Spottings; Techniques; Tissue Sample; Tissues; Training; Trypsin; Ultracentrifugation; United States National Institutes of Health; Up-Regulation; Work; base; falls; insight; multidisciplinary; novel; social; subcutaneous; tool; volunteer

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. Protein deposition as amyloid is the basis of diseases that, overall, have an enormous social and medical impact. More than 21 different proteins are known to be causative agents. In the systemic forms, amyloid deposition is associated with dysfunction of vital organs, and molecular typing of the deposits is necessary for diagnosis and treatment. The traditional diagnostic approach is multidisciplinary, but sometimes fails to identify the correct type. A proteomic approach could help in the diagnosis, through the direct molecular characterization of fibrillar proteins in tissues, and cast insights into the mechanisms of tissue damage. We are working to characterize amyloid deposits in abdominal subcutaneous fat from patients with systemic amyloidosis using 2D-PAGE followed by MALDI-TOF MS and peptide mass fingerprinting. Fat tissue samples were obtained from individuals affected by various forms of systemic amyloidoses. Samples from unaffected volunteers were used as normal controls. Protein was extracted from fat tissue by homogenization directly in IEF buffer followed by ultracentrifugation to clear debris and delipidate samples. Samples were then subjected to 2D-PAGE analysis and Coomassie or silver staining. Protein spots were imaged and quantitated using PDQuest" software. Spots indicating differentially expressed proteins are being excised and subjected to in-gel digestion by trypsin or another protease. Peptides are eluted, de-salted and analyzed by MALDI-TOF MS or by LC-MS and MS/MS. Spectra are analyzed with MoverZ (M/Z") or MassLynx" and ProteinLynx" software, and peptide mass fingerprinting analysis utilizes MASCOT and/or BUPID. A rapid methodology to prepare sample for high-grade 2D-PAGE analysis from fat tissue has been developed. Fat tissues from unaffected (non-amyloid) volunteers were used to generate 2D reference maps for comparison with those generated from patients affected by systemic amyloidosis. Preliminary results arising from the comparison of these maps indicate that significant differences exist. The observations surrounding these differences fall into three categories. (1) New spots appear in the region of the gel consistent with the location where an amyloidogenic protein would typically be found. (2) There is apparent upregulation of some proteins in 2D maps from patient samples compared to those originating from unaffected volunteers. (3) New spots are observed in patient maps that are absent in the non-amyloid maps, suggesting the appearance of novel proteins. These new spots often appear in "trains" suggesting the presence of modified forms of the same protein. Identifation of the proteins responsible for these differences is being accomplished using in-gel protease digestion and a variety of MS techniques. The use of 2D-PAGE as a tool to highlight the differences between diseased and normal states is well known. Our proteomic approach to the analysis of fat tissues from patients for whom amyloid disease is suspected that should be able to provide a reliable diagnosis. This approach is practical and feasible, given that fat aspirates of potential amyloid patients are routinely acquired for histological analysis. Ongoing analyses may provide identification of new aspects of the disease mechanisms, including the involvement of novel proteins and protein post-translational modifications.

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。 淀粉样蛋白沉积是疾病的基础，总体而言，具有巨大的社会和医学影响。已知有超过21种不同的蛋白质是致病因子。在系统性形式中，淀粉样蛋白沉积与重要器官的功能障碍相关，并且沉积物的分子分型对于诊断和治疗是必要的。传统的诊断方法是多学科的，但有时无法识别正确的类型。蛋白质组学方法可以通过组织中纤维蛋白的直接分子表征来帮助诊断，并深入了解组织损伤的机制。我们正在研究系统性淀粉样变性患者腹部皮下脂肪中淀粉样沉积物的特征，采用2D-PAGE，然后进行MALDI-TOF MS和肽质量指纹图谱分析。 脂肪组织样品从受各种形式的系统性淀粉样变性影响的个体获得。未受影响志愿者的样本用作正常对照。通过直接在IEF缓冲液中均质化，然后超离心以清除碎片和脱脂样品，从脂肪组织中提取蛋白质。然后对样品进行2D-PAGE分析和考马斯或银染色。 使用PDQuest软件对蛋白质点进行成像和定量。切下指示差异表达的蛋白质的点，并通过胰蛋白酶或另一种蛋白酶进行凝胶内消化。将肽洗脱、脱盐并通过MALDI-TOF MS或通过LC-MS和MS/MS分析。用MoverZ（M/Z "）或MassLynx"和ProteinLynx "软件分析光谱，并且肽质量指纹分析利用MASCOT和/或BUPID。建立了一种快速制备脂肪组织高纯度2D-PAGE分析样品的方法。来自未受影响的（非淀粉样蛋白）志愿者的脂肪组织用于生成2D参考图，以与来自受系统性淀粉样变性影响的患者的脂肪组织进行比较。 对这些地图进行比较的初步结果表明，存在着重大差异。围绕这些差异的观察分为三类。(1)新的斑点出现在与淀粉样蛋白通常被发现的位置一致的凝胶区域中。（2）与来自未受影响的志愿者的那些相比，来自患者样品的2D图谱中的一些蛋白质存在明显的上调。(3)在患者图谱中观察到非淀粉样蛋白图谱中不存在的新斑点，表明新蛋白质的出现。这些新的斑点经常出现在“火车”上，表明存在相同蛋白质的修饰形式。负责这些差异的蛋白质的鉴定正在使用凝胶内蛋白酶消化和各种MS技术来完成。使用2D-PAGE作为突出疾病和正常状态之间的差异的工具是众所周知的。我们的蛋白质组学方法对怀疑淀粉样疾病的患者的脂肪组织进行分析，应该能够提供可靠的诊断。这种方法是实用和可行的，因为潜在的淀粉样蛋白患者的脂肪抽吸物是常规采集用于组织学分析的。 正在进行的分析可能提供疾病机制的新方面的鉴定，包括新蛋白质和蛋白质翻译后修饰的参与。