Lavatelli Francesca, Perlman David H, Spencer Brian, Prokaeva Tatiana, McComb Mark E, Théberge Roger, Connors Lawreen H, Bellotti Vittorio, Seldin David C, Merlini Giampaolo, Skinner Martha, Costello Catherine E
Amyloid Treatment and Research Program, Boston, Massachusetts, USA.
Mol Cell Proteomics. 2008 Aug;7(8):1570-83. doi: 10.1074/mcp.M700545-MCP200. Epub 2008 May 12.
In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of alphaB-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration.
在系统性淀粉样变性中，蛋白质以淀粉样蛋白形式广泛沉积会导致严重的器官功能障碍。为设计合适的治疗策略，有必要区分不同形式的淀粉样蛋白。我们开发了一种蛋白质组学方法，利用二维聚丙烯酰胺凝胶电泳，随后进行基质辅助激光解吸/电离质谱分析和肽质量指纹识别，以直接表征通过细针穿刺从被诊断为免疫球蛋白轻链型或转甲状腺素蛋白型淀粉样变性的患者腹部皮下脂肪中获取的淀粉样沉积物。在对照组与患者之间以及两类患者之间，观察到脂肪组织二维凝胶蛋白质组存在显著差异，患者标本中有独特的额外斑点，这些斑点可被确定为全长和截短形式的淀粉样蛋白生成蛋白。在转甲状腺素蛋白突变的杂合子患者中，从同一蛋白质斑点中分离出的野生型肽和含有淀粉样转甲状腺素蛋白变体的肽数量大致相等，这表明两种类型的肽都掺入了沉积物中。此外，患者样本中出现了与淀粉样蛋白生成蛋白无关的新斑点；其中一些被鉴定为血清淀粉样蛋白P和载脂蛋白E的异构体，此前已有描述称这些蛋白质与淀粉样沉积物有关。最后，观察到驻留脂肪蛋白的正常表达模式发生了变化，如αB-晶状体蛋白、过氧化物还原酶6和醛糖酮还原酶I的下调，这显然与淀粉样蛋白的存在有关，尽管它们的水平与淀粉样蛋白沉积程度并不严格相关。这种蛋白质组学方法不仅提供了一种检测和明确鉴定淀粉样变性患者腹部皮下脂肪抽吸物中沉积物类型的方法，还可能通过识别与淀粉样蛋白浸润相关的新蛋白质或蛋白质翻译后修饰，为疾病机制提供新的见解。
