■淀粉样变症是由器官和组织中各种类型的血清蛋白沉积引起的危及生命的疾病。了解所涉及的蛋白质类型是正确诊断和个性化医疗的基础。虽然经典方法使用免疫组织化学,近年来,激光显微解剖,其次是高分辨率LC-MS/MS,已被证明具有优异的诊断灵敏度和特异性。这些技术,然而,仅在主要参考蛋白质组学中心可用。
■使用低分辨率质谱和无激光显微解剖(LMD)进行临床淀粉样蛋白分型,我们开发了一种靶向蛋白质组学方法,用于测定两种经常遇到的淀粉样蛋白(即,κ和-λ免疫球蛋白轻链和甲状腺素运载蛋白(TTR)和特定参考蛋白(即,肌动蛋白(A)用于心肌组织,或皮下脂肪组织的脂肪酸结合蛋白4(FBP4))在组织学标本中。
■小组织碎片和/或组织切片被消化以产生随后被还原的蛋白质混合物,烷基化和胰蛋白酶化以获得肽混合物。SPE纯化和LC分离后,蛋白型肽通过它们的MRM转换来检测。
■该方法对淀粉样蛋白蛋白蛋白型肽显示出高特异性和敏感性。对于TTR,心肌组织(CMT)的LOD为1.0、0.1、0.2皮摩尔,皮下脂肪组织(SAT)的LOD为0.1、0.2、0.5皮摩尔,κ-,和λ-LC蛋白,分别。淀粉样蛋白与组织特异性蛋白信号比与临床样品中淀粉样蛋白沉积物的存在相关。
■这种靶向蛋白质组学方法能够对淀粉样变性受影响的组织进行敏感和特异性的区分,以用于临床研究。
UNASSIGNED: Amyloidosis is a life threatening disease caused by deposition of various types of blood serum proteins in organs and tissues. Knowing the type of protein involved is the basis of a correct diagnosis and personalized medical treatment. While the classical approach uses immunohistochemistry, in recent years, laser micro-dissection, followed by high resolution LC-MS/MS, has been shown to provide superior diagnostic sensitivity and specificity. This techniques, however, is only available at major reference proteomics centers.
UNASSIGNED: To perform clinical amyloid protein typing using low-resolution mass spectrometry and no laser micro dissection (LMD), we developed a targeted proteomics approach for the determination of both frequently encountered amyloid proteins (i.e., κ and -λ immunoglobulin light chains and transthyretin (TTR)) and specific reference proteins (i.e., actin (A) for cardiac muscle tissue, or fatty acid binding protein 4 (FBP4) for subcutaneous adipose tissue) in histologic specimens.
UNASSIGNED: Small tissue fragments and/or histological sections were digested to yield a protein mixture that was subsequently reduced, alkylated and trypsinized to obtain a peptide mixture. After SPE purification and LC separation, proteotypic peptides were detected by their MRM transitions.
UNASSIGNED: The method showed high specificity and sensitivity for amyloid protein proteotypic peptides. LODs were 1.0, 0.1, 0.2 picomoles in cardiac muscle tissue (CMT) and 0.1, 0.2, 0.5 picomoles in subcutaneous adipose tissue (SAT) for TTR, κ-, and λ-LC proteins, respectively. Amyloid to tissue-specific protein signal ratios correlated with the presence of amyloid deposits in clinical samples.
UNASSIGNED: This targeted proteomics approach enables sensitive and specific discrimination of amyloidosis affected tissues for the purpose of clinical research.