Bottom-up mass spectrometry–based proteomics relies on protein digestion and peptide purification. The application of such methods to broadly available clinical samples such as formalin-fixed and paraffin-embedded (FFPE) tissues requires reversal of chemical crosslinking and the removal of reagents that are incompatible with mass spectrometry. Here, we describe in detail a protocol that combines tissue disruption by ultrasonication, heat-induced antigen retrieval and two alternative methods for efficient detergent removal to enable quantitative proteomic analysis of limited amounts of FFPE material. To show the applicability of our approach, we used hepatocellular carcinoma (HCC) as a model system. By combining the described protocol with laser-capture microdissection, we were able to quantify the intra-tumor heterogeneity of a tumor specimen on the proteome level using a single slide with tissue of 10-µm thickness. We also demonstrate broader applicability to other tissues, including human gallbladder and heart. The procedure described in this protocol can be completed within 8 d.

基于自下而上质谱的蛋白质组学依赖于蛋白质消化和肽纯化。将此类方法应用于广泛可用的临床样本，如福尔马林固定和石蜡包埋 (FFPE) 组织，需要逆转化学交联并去除与质谱法不相容的试剂。在这里，我们详细描述了一个协议，该协议结合了超声波处理组织破坏、热诱导抗原修复和两种有效去除洗涤剂的替代方法，从而能够对有限量的 FFPE 材料进行定量蛋白质组学分析。为了展示我们方法的适用性，我们使用肝细胞癌 (HCC) 作为模型系统。通过将描述的协议与激光捕获显微切割相结合，我们能够使用具有 10 µm 厚度组织的单个载玻片在蛋白质组水平上量化肿瘤标本的肿瘤内异质性。我们还展示了对其他组织的更广泛适用性，包括人类胆囊和心脏。本协议中描述的程序可在 8 天内完成。