VIB-UGent Center for Medical Biotechnology, Ghent, Belgium.
Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
Methods Mol Biol. 2023;2718:213-233. doi: 10.1007/978-1-0716-3457-8_12.
Amyloidosis is a group of rare pathologies characterized by abnormal folding and deposition of susceptible proteins in tissues and organs. Diagnosis of amyloidosis often relies on immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) patient samples; however, dependency on antibodies for protein staining is one of the major pitfalls of this approach, especially for the detection of rare amyloidosis types. In recent years, mass spectrometry-based proteomics has emerged as a promising alternative for adequate detection and amyloid typing, despite the fact that preparing FFPE samples for proteomics remains a challenging task. Major hurdles are removal of formalin-induced protein cross-links and water-insoluble paraffin prior to mass spectrometry analysis. With the recent development of the suspension trapping protocol, enabling the use of high concentrations of SDS, these obstacles can be overcome. In this chapter, we describe the implementation of suspension trapping for FFPE sample processing and its application to analyze human amyloidosis samples, comparing a standard procedure with probe sonication with a more advanced workflow based on ultrasonication.
淀粉样变性是一组由组织和器官中易感性蛋白质异常折叠和沉积引起的罕见病理学。淀粉样变性的诊断通常依赖于福尔马林固定石蜡包埋（FFPE）患者样本的免疫组织化学；然而，这种方法的主要缺陷之一是依赖于抗体进行蛋白质染色，尤其是对于检测罕见的淀粉样变性类型。近年来，基于质谱的蛋白质组学已成为一种很有前途的替代方法，可用于充分检测和淀粉样蛋白分型，尽管为蛋白质组学准备 FFPE 样本仍然是一项具有挑战性的任务。主要障碍是在进行质谱分析之前去除甲醛诱导的蛋白质交联和不溶于水的石蜡。随着悬浮捕获方案的最新发展，能够使用高浓度 SDS，这些障碍可以被克服。在这一章中，我们描述了悬浮捕获在 FFPE 样本处理中的实施及其在分析人类淀粉样变性样本中的应用，将标准程序与探针超声处理与基于超声处理的更先进的工作流程进行了比较。
