Addis Maria Filippa, Tanca Alessandro, Pagnozzi Daniela, Crobu Salvatore, Fanciulli Giuseppe, Cossu-Rocca Paolo, Uzzau Sergio
Porto Conte Ricerche Srl, Tramariglio, Alghero, Sassari, Italy.
Proteomics. 2009 Aug;9(15):3815-23. doi: 10.1002/pmic.200800971.
A wealth of information on proteins involved in many aspects of disease is encased within formalin-fixed paraffin-embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this "hidden treasure" is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full-length proteins. Here, we describe an optimised method for extraction of full-length proteins from FFPE tissues. This method builds on the classical "antigen retrieval" technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3 microg and 86.8 microg of proteins were obtained per 80 mm(2) tissue slice of formalin-fixed paraffin-embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS-PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC-nanoESI-Q-TOF MS of FFPE proteins resolved by SDS-PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported.
世界各地医院保存的福尔马林固定石蜡包埋（FFPE）组织库中蕴藏着大量与疾病诸多方面相关的蛋白质信息。最近，通过两种主要提取策略的应用，人们正在积极探寻获取这些“隐藏宝藏”的方法：一是用胰蛋白酶消化缠结的蛋白质基质以生成胰蛋白酶肽段，二是进行去交联并提取全长蛋白质。在此，我们描述一种从FFPE组织中提取全长蛋白质的优化方法。该方法基于用于免疫组织化学的经典“抗原修复”技术，能够产生产量提高且可重复的蛋白质提取物。在模型动物组织中，每80平方毫米福尔马林固定石蜡包埋的骨骼肌和肝脏组织切片分别平均获得16.3微克和86.8微克蛋白质。用这种方法产生的蛋白质提取物可用于采用多种技术对蛋白质组进行可重复的研究。本文展示并讨论了通过SDS-PAGE、western免疫印迹、蛋白质芯片、ELISA，以及最重要的是对经SDS-PAGE分离的FFPE蛋白质进行的纳升液相色谱-纳升电喷雾-四极杆-飞行时间质谱（nanoHPLC-nanoESI-Q-TOF MS）所获得的结果。还报告了对福尔马林固定和交联逆转对蛋白质造成的修饰程度及其对质谱结果质量的影响的评估。
