Fowler Carol B, O'Leary Timothy J, Mason Jeffrey T
Department of Biophysics, Armed Forces Institute of Pathology, Rockville, MD 20850, USA.
Lab Invest. 2008 Jul;88(7):785-91. doi: 10.1038/labinvest.2008.43. Epub 2008 May 19.
Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60 degrees C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology. Following histological processing, proteins could be extracted from the tissue surrogates by combining heat, detergent, and a protein denaturant. However, gel electrophoresis revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins. This suggested that during the subsequent steps of tissue processing protein-formaldehyde adducts undergo further modifications that are not observed in aqueous proteins. As a first step toward understanding these additional modifications we have performed a comparative evaluation of RNase A following fixation in buffered formaldehyde alone and after subsequent dehydration in 100% ethanol by combining gel electrophoresis, chemical modification, and circular dichroism spectroscopic studies. Our results reveal that ethanol-induced rearrangement of the conformation of fixed RNase A leads to protein aggregation through the formation of large geometrically compatible hydrophobic beta-sheets that are likely stabilized by formaldehyde cross-links, hydrogen bonds, and van der Waals interactions. It requires substantial energy to reverse the formaldehyde cross-links within these sheets and regenerate protein monomers free of formaldehyde modifications. Accordingly, the ethanol-dehydration step in tissue histology may be important in confounding the successful recovery of proteins from FFPE tissues for immunohistochemical and proteomic analysis.
了解甲醛固定及后续组织处理过程中蛋白质修饰的化学机制，对于开发改进的免疫组织化学抗原修复方法以及从福尔马林固定、石蜡包埋（FFPE）组织中提取蛋白质进行蛋白质组学分析至关重要。我们最初对10%缓冲福尔马林溶液中的单一蛋白质（如牛胰核糖核酸酶A（RNase A））进行的研究表明，去除过量甲醛后，通过在pH 4条件下于60℃加热30分钟，可回收具有正常免疫反应性的单体RNase A。接下来，我们研究了组织替代物，它们是固定蛋白质的明胶样栓块，具有足够的物理完整性，可采用常规组织组织学方法进行处理。经过组织学处理后，可通过加热、去污剂和蛋白质变性剂相结合的方式从组织替代物中提取蛋白质。然而，凝胶电泳显示替代物提取物中含有单体和多聚体蛋白质的混合物。这表明在组织处理的后续步骤中，蛋白质 - 甲醛加合物会发生进一步修饰，而这在水性蛋白质中未观察到。作为理解这些额外修饰的第一步，我们通过结合凝胶电泳、化学修饰和圆二色光谱研究，对单独在缓冲甲醛中固定后以及随后在100%乙醇中脱水后的RNase A进行了比较评估。我们的结果表明，乙醇诱导固定RNase A构象重排，通过形成大的几何相容疏水β - 折叠导致蛋白质聚集，这些β - 折叠可能通过甲醛交联、氢键和范德华相互作用得以稳定。要逆转这些折叠内的甲醛交联并再生无甲醛修饰的蛋白质单体需要大量能量。因此，组织组织学中的乙醇脱水步骤可能对从FFPE组织中成功回收用于免疫组织化学和蛋白质组学分析的蛋白质具有重要影响。
