Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting
Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting
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DOI：
10.1002/path.2504
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发表时间：
2009-03-01
影响因子：
7.3
通讯作者：
Banks, Rosamonde E.
中科院分区：
文献类型：
--
作者：
Nirmalan, Niroshini J.;Harnden, Patricia;Banks, Rosamonde E.
有效的甲醛交联逆转策略的发展将使病理学部门的大量诊断组织档案适合于前瞻性和回顾性转化研究，特别是在生物标志物驱动的蛋白质组学研究中。热诱导抗原修复策略（HIAR）已经实现了不同程度的交联逆转，可能使档案组织用于其目前免疫组织化学（IHC）范围之外的蛋白质组学应用。虽然迄今为止取得的大多数成功都是基于使用鸟枪蛋白质组学方法检索胰蛋白酶肽片段，但从存档组织中提取全长、非降解、免疫反应性蛋白质的尝试已被证明具有挑战性。我们已经开发了一种新的热诱导抗原修复策略，使用SDS-含有Laemmli缓冲液，从福尔马林固定的组织中有效地回收完整的蛋白质，用于随后的Western印迹分析。介绍了冷冻组织和当前领先方法的提取效率的方案优化和比较。在来自10名患者的匹配的肿瘤/正常、冷冻/FFPE肾组织样本的队列中进行方法学的定量验证，通过蛋白质印迹法探测已知在肾癌中差异表达的七种蛋白质的选定组。我们的数据显示，该方案能够有效提取非降解的全长免疫反应性蛋白质，对于大多数测试的蛋白质组，肿瘤与正常差异表达谱与匹配的冷冻组织对照相当（秩相关，r = 0.7292，p < 1.825e-09）。然而，在某些膜蛋白的提取效率中观察到的变异性强调了对来自该蛋白质子集的定量数据进行谨慎解释的必要性。该方法提供了一个可行的，具有成本效益的定量选择，通过一系列的临床样本，从现有的诊断档案，验证潜在的生物标志物面板，前提是该方法的验证是首先进行的特定蛋白质的研究。版权所有（C）2008大不列颠和爱尔兰病理学会。由John Wiley & Sons有限公司出版
The development of efficient formaldehyde cross-link reversal strategies will make the vast diagnostic tissue archives of pathology departments amenable to prospective and retrospective translational research, particularly in biomarker-driven proteomic investigations. Heat-induced antigen retrieval strategies (HIARs) have achieved varying degrees of cross-link reversal, potentially enabling archival tissue usage for proteomic applications outside its current remit of immunohistochemistry (IHC). While most successes achieved so far have been based on retrieving tryptic peptide fragments using shot-gun proteomic approaches, attempts at extracting full-length, non-degraded, immunoreactive proteins from archival tissue have proved challenging. We have developed a novel heat-induced antigen retrieval strategy using SDS-containing Laemmli buffer for efficient intact protein recovery from formalin-fixed tissues for subsequent analysis by western blotting. Protocol optimization and comparison of extraction efficacies with frozen tissues and current leader methodology is presented. Quantitative validation of methodology was carried out in a cohort of matched tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting for a selected panel of seven proteins known to be differentially expressed in renal cancer. Our data show that the protocol enables efficient extraction of non-degraded, full-length, immunoreactive protein, with tumour versus normal differential expression profiles for a majority of the panel of proteins tested being comparable to matched frozen tissue controls (rank correlation, r = 0.7292, p < 1.825e-09). However, the variability observed in extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation of quantitative data from this subset of proteins. The method provides a viable, cost-effective quantitative option for the validation of potential biomarker panels through a range of clinical samples from existing diagnostic archives, provided that validation of the method is first carried out for the specific proteins under study. Copyright (C) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.