Collaud Stéphane, Wiedl Thomas, Cattaneo Elisa, Soltermann Alex, Hillinger Sven, Weder Walter, Arni Stephan
Department of Thoracic Surgery, University Hospital Zurich, Switzerland.
J Biomol Tech. 2010 Apr;21(1):25-8.
Laser-capture microdissection (LCM) enables the selection of a specific and pure cell population from a heterogenous tissue such as tumors. Activity-based protein profiling/profile (ABPP) is a chemical technology using enzyme-specific active site-directed probes to read out the functional state of many enzymes directly in any proteome. The aim of this work was to assess the compatibility of LCM with downstream ABPP for serine hydrolase (SH) in human lung adenocarcinoma. Fresh frozen lung adenocarcinoma tissue was stained with hematoxylin, toluidine blue, or methyl green (MG). Proteome from stained tissue was labeled further with SH-directed probes, and ABPPs were determined on a one-dimensional gel-based approach. This allowed us to assess the impact of staining procedures on their ABPPs. The effect of the LCM process on ABPPs was assessed furthermore using MG-stained lung adenocarcinoma tissue. The staining procedures led to strong changes in ABPPs. However, MG staining seemed the most compatible with downstream ABPP. MG-stained, laser-captured, microdissected tissue showed additional change in profiles as a result of the denaturing property of extraction buffer but not to the microdissection process itself. LCM staining procedures but not microdissection per se interfered with downstream ABPP and led to a strong change in ABPPs of SHs in human lung adenocarcinoma.
激光捕获显微切割（LCM）能够从诸如肿瘤等异质性组织中挑选出特定且纯净的细胞群体。基于活性的蛋白质谱分析（ABPP）是一种化学技术，它使用酶特异性活性位点导向探针直接在任何蛋白质组中读出多种酶的功能状态。这项工作的目的是评估LCM与人类肺腺癌中丝氨酸水解酶（SH）的下游ABPP的兼容性。将新鲜冷冻的肺腺癌组织用苏木精、甲苯胺蓝或甲基绿（MG）染色。用SH导向探针进一步标记染色组织的蛋白质组，并通过一维凝胶法测定ABPP。这使我们能够评估染色程序对其ABPP的影响。此外，使用MG染色的肺腺癌组织评估LCM过程对ABPP的影响。染色程序导致ABPP发生强烈变化。然而，MG染色似乎与下游ABPP最兼容。MG染色、激光捕获、显微切割的组织由于提取缓冲液的变性特性而在谱图上显示出额外的变化，但不是由于显微切割过程本身。LCM染色程序而非显微切割本身干扰了下游ABPP，并导致人类肺腺癌中SH的ABPP发生强烈变化。
