PROTEOMIC APPROACH TO THE STUDY OF SYSTEMIC AMYLOIDOSIS

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。 作为淀粉样蛋白的蛋白质沉积是疾病的基础，总的来说，这些疾病具有巨大的社会和医学影响。目前已知有超过21种不同的蛋白质是致病因子。在全身类型中，淀粉样蛋白沉积与重要器官的功能障碍有关，对沉积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直接对组织中的纤维蛋白进行分子表征来帮助诊断，并对组织损伤的机制有深入的了解。我们正致力于使用2D-PAGE、MALDI-TOF、MS和多肽质量指纹图谱来表征系统性淀粉样变性患者腹部皮下脂肪中的淀粉样沉积。脂肪组织样本取自各种形式的系统性淀粉样变性患者。健康志愿者的样本作为正常对照。脂肪组织中的蛋白质直接在IEF缓冲液中匀浆提取，然后超速离心法清除碎屑和脱脂样品。然后对样品进行2D-PAGE分析和考马斯亮蓝或银染。用“PDQuest”软件对蛋白质斑点进行成像和定量。切除差异表达蛋白质的斑点，用胰酶或其他酶进行凝胶内消化。用MALDI-TOF MS或LC-MS和MS/MS洗脱、脱盐和分析多肽。用MoverZ(M/Z“)或MassLynx”和ProteinLynx“软件分析波谱，利用吉祥物和/或BUPID进行多肽质量指纹图谱分析。发展了一种从脂肪组织中快速制备样品以进行高级2D-PAGE分析的方法。来自未受影响的(非淀粉样蛋白)志愿者的脂肪组织被用来生成2D参考地图，以与系统性淀粉样变性患者生成的参考地图进行比较。对这些地图进行比较的初步结果表明，存在显著差异。围绕这些差异的观察可分为三类。(1)在凝胶的区域出现新的斑点，该区域与通常发现淀粉样蛋白的位置一致。(2)与来自健康志愿者的蛋白质相比，来自患者样本的2D图谱中的某些蛋白质有明显的上调。(3)在患者图谱中观察到非淀粉样蛋白图谱中没有的新斑点，这表明出现了新的蛋白质。这些新斑点经常出现在“列车”中，表明存在相同蛋白质的修饰形式。对造成这些差异的蛋白质的鉴定是使用凝胶内蛋白酶消化和各种MS技术来完成的。使用2D-PAGE作为一种工具来突出疾病和正常状态之间的差异是众所周知的。我们对疑似淀粉样变性患者的脂肪组织进行蛋白质组学分析，应该能够提供可靠的诊断。这种方法是实用和可行的，因为潜在的淀粉样变患者的脂肪抽吸物是常规获取的，用于组织学分析。正在进行的分析可能提供疾病机制的新方面的鉴定，包括参与新的蛋白质和蛋白质翻译后修饰。

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.