Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.
Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.
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DOI：
10.1371/journal.pone.0014253
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发表时间：
2010-12-08
期刊：
影响因子：
3.7
通讯作者：
Mason JT
中科院分区：
文献类型：
--
作者：
Fowler CB;Chesnick IE;Moore CD;O'Leary TJ;Mason JT
福尔马林固定的石蜡包埋（FFPE）组织的蛋白质组学研究的挫折，无法从档案组织中提取蛋白质的形式，适合通过2-D凝胶电泳或质谱分析。这种无能是由于难以逆转福尔马林诱导的蛋白加合物和FFPE组织内的交联。我们以前曾报道过使用高的静水压力作为一种方法，从鸡蛋白溶菌酶组织替代品，模型系统开发研究福尔马林固定和组织化学处理的有效蛋白质回收。在这项研究中，我们证明了提高静水压力作为一种有效的方法，从FFPE小鼠肝组织和复杂的多蛋白FFPE组织替代物的回收蛋白质的方法，该组织替代物由鸡蛋清溶菌酶，牛碳酸酐酶，牛核糖核酸酶A，牛血清白蛋白和马肌红蛋白（55∶15∶ 15∶10∶ 5wt%）组成。在升高的压力下检索的FFPE组织替代物的质谱分析显示，低丰度和高丰度蛋白质都被鉴定为具有与甲醛处理之前的替代物混合物相当的序列覆盖率。相比之下，非压力提取的组织替代样品产生很少的阳性和许多错误的肽鉴定。用可溶性福尔马林处理的牛核糖核酸酶A的研究表明，压力适度抑制甲醛诱导的蛋白质交联的逆转（水解）速率。动态光散射研究表明，提高静水压力和热量有利于恢复蛋白质的甲醛加合物和交联，促进蛋白质展开和水化与伴随的平均大小的蛋白质聚集体的减少。这些研究表明，提高静水压力处理是一种有前途的方法，用于改善从FFPE组织中回收的蛋白质的形式适合于蛋白质组学分析。
Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.
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影响因子：
3.7
作者：
Machida K;Eschrich S;Li J;Bai Y;Koomen J;Mayer BJ;Haura EB
通讯作者：
Haura EB
DOI：
10.1007/978-1-59745-324-0_13
发表时间：
2010-01-01
期刊：
IMMUNOCYTOCHEMICAL METHODS AND PROTOCOLS, THIRD EDITION
影响因子：
--
作者：
Bratthauer, Gary L.
通讯作者：
Bratthauer, Gary L.
影响因子：
3.2
作者：
Guo, Tong;Wang, Weijie;Balgley, Brian M.
通讯作者：
Balgley, Brian M.
影响因子：
6.2
作者：
Jou, Yu-Jen;Lin, Chia-Der;Lin, Cheng-Wen
通讯作者：
Lin, Cheng-Wen
影响因子：
3.3
作者：
Nishimura, Toshihide;Nomura, Masaharu;Kato, Harubumi
通讯作者：
Kato, Harubumi