Background

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.

Objective

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.

Method

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.

Results

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.

Conclusions

This targeted proteomics approach enables sensitive and specific discrimination of amyloidosis affected tissues for the purpose of clinical research.

中文翻译：

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淀粉样变性分型的靶向蛋白质组学方法

 背景

淀粉样变性是由各种类型的血清蛋白在器官和组织中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，但近年来，激光显微切割和高分辨率 LC-MS/MS 已被证明可以提供卓越的诊断灵敏度和特异性。然而，这种技术仅在主要的参考蛋白质组学中心可用。

 客观的

为了使用低分辨率质谱法和非激光显微解剖 (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，用于测定常见的淀粉样蛋白（即 κ 和 -λ 免疫球蛋白轻链和转甲状腺素蛋白 (TTR)） )) 和组织学标本中的特定参考蛋白（即心肌组织的肌动蛋白 (A) 或皮下脂肪组织的脂肪酸结合蛋白 4 (FBP4)）。

 方法

消化小组织碎片和/或组织切片以产生蛋白质混合物，随后将其还原、烷基化和胰蛋白酶化以获得肽混合物。经过 SPE 纯化和 LC 分离后，通过 MRM 离子对检测蛋白型肽。

 结果

该方法对淀粉样蛋白蛋白肽表现出高特异性和敏感性。 TTR、κ-和 λ-LC 蛋白在心肌组织 (CMT) 中的 LOD 分别为 1.0、0.1、0.2 皮摩尔，在皮下脂肪组织 (SAT) 中分别为 0.1、0.2、0.5 皮摩尔。淀粉样蛋白与组织特异性蛋白信号比与临床样本中淀粉样蛋白沉积物的存在相关。

 结论

这种有针对性的蛋白质组学方法能够敏感且特异性地区分淀粉样变性影响的组织，以用于临床研究。