Fowler Carol B, Cunningham Robert E, Waybright Timothy J, Blonder Josip, Veenstra Timothy D, O'Leary Timothy J, Mason Jeffrey T
Department of Biophysics, Armed Forces Institute of Pathology, Rockville, MD 20850, USA.
Lab Invest. 2008 Feb;88(2):185-95. doi: 10.1038/labinvest.3700708. Epub 2007 Dec 24.
High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of 'tissue surrogates' as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates. Reversal of formaldehyde-induced protein adducts and crosslinks was observed when lysozyme tissue surrogates were extracted at 45 000 psi and 80-100 degrees C in Tris buffers containing 2% sodium dodecyl sulfate and 0.2 M glycine at pH 4. These conditions also produced peptides resulting from acid-catalyzed aspartic acid cleavage. Additives such as trimethylamine N-oxide or copper (II) chloride decreased the total percentage of these aspartic acid cleavage products, while maintaining efficient reversal of intermolecular crosslinks in the FFPE tissue surrogates. Mass spectrometry analysis of the recovered lysozyme yielded 70% sequence coverage, correctly identified all formaldehyde-reactive amino acids, and demonstrated hydrolysis at all of the expected trypsin cleavage sites. This study demonstrates that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues for proteomic analysis.
对福尔马林固定、石蜡包埋（FFPE）组织进行的高通量蛋白质组学研究一直受到从存档组织中提取蛋白质的低效方法以及对甲醛诱导的蛋白质修饰了解不全面的阻碍。我们之前报道了一种形成“组织替代物”的方法，作为研究福尔马林固定、组织化学处理以及从FFPE组织中回收蛋白质的模型。在本研究中，我们展示了使用高静水压作为从FFPE组织替代物中高效回收蛋白质的方法。当溶菌酶组织替代物在含有2%十二烷基硫酸钠和0.2 M甘氨酸（pH 4）的Tris缓冲液中于45000 psi和80 - 100摄氏度下提取时，观察到甲醛诱导的蛋白质加合物和交联的逆转。这些条件还产生了由酸催化天冬氨酸裂解产生的肽段。诸如氧化三甲胺或氯化铜（II）等添加剂降低了这些天冬氨酸裂解产物的总百分比，同时保持了FFPE组织替代物中分子间交联的有效逆转。对回收的溶菌酶进行质谱分析，序列覆盖率达到70%，正确识别了所有甲醛反应性氨基酸，并证明在所有预期的胰蛋白酶裂解位点均发生了水解。本研究表明，高静水压处理是一种有前景的方法，可用于改善从FFPE组织中回收蛋白质以进行蛋白质组学分析。
