Recovery of Protein from Formalin-Fixed Tissues

描述（由申请人提供）：了解癌症分子发病机制最有前途的方法之一是使用高通量分子生物学和蛋白质组学方法将遗传变化（如突变或改变的基因表达）与转移、治疗结果和生存联系起来。在癌症中，从最初的诊断和治疗到转移的出现时间较长，必须通过福尔马林固定石蜡包埋（FFPE）组织获得临床相关性。然而，大规模的多重技术，如蛋白质组学分析、基因表达序列分析和使用FFPE组织的基因芯片方法，先前被证明是不成功的。我们研究项目的长期目标是通过使用高通量蛋白质组学和分子生物学筛选方法来识别癌症的分子和遗传特征，从而改善公众健康。本提案的目的是利用组织替代物来鉴定正常组织学组织加工过程中发生的甲醛诱导的蛋白质化学修饰，并开发逆转这些修饰的方法。组织替代物是高度浓缩的蛋白质溶液，在甲醛诱导的分子间交联后形成组织样的塞。这些塞可以像正常组织一样进行组织学处理。我们的中心假设是，在适当的溶剂化条件下，通过精心设计的一系列加热、透析、补液和蛋白质复性步骤，可以依次逆转在组织学组织处理过程中形成的甲醛加合物和交联物。我们在强有力的初步数据的基础上提出了这一假设，这些数据表明，甲醛诱导的蛋白质化学变化的逆转在水溶液中相对容易，但对于在有机溶剂存在下脱水的组织需要不同的方法。这些研究的基本原理是，它们的成功完成将为FFPE组织的高通量蛋白质组学筛选提供基础。这将改善癌症的诊断、治疗和预防的实际干预措施，并将促进治疗剂的发展。我们的研究是创新的，因为我们已经开发了一种新的模型系统（组织替代品），非常适合于识别在组织学处理过程中发生的甲醛诱导的蛋白质修饰。在这个项目完成后，我们期望对组织组织学过程中发生的蛋白质修饰有一个全面的了解，以及最佳逆转这些修饰的方法。这些知识应该导致使用FFPE组织进行蛋白质组学分析的能力。

DESCRIPTION (provided by applicant): One of the most promising approaches for understanding the molecular pathogenesis of cancer is to relate genetic changes, such as mutation or altered gene expression, to metastasis, treatment outcome, and survival, using high-throughput molecular biologic and proteomic methods. In cancers where the time between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations must be obtained with formalin-fixed paraffin-embedded (FFPE) tissues. However, large-scale multiplex techniques, such as proteomic analysis, serial analysis of gene expression, and gene chip methods using FFPE tissue have previously proven unsuccessful. The long-term goal of our research program is to improve public health by using high-throughput proteomic and molecular biologic screening methods to identify the molecular and genetic signatures of cancer. The objective of this proposal is to employ tissue surrogates to identify the formaldehyde-induced chemical modifications to proteins that occur during normal histologic tissue processing and to develop methods to reverse these modifications. Tissue surrogates are highly concentrated solutions of proteins that form tissue-like plugs following formaldehyde-induced intermolecular crosslinking. These plugs can be histologically processed like normal tissue. Our central hypothesis is that formaldehyde adducts and cross-links formed during histologic tissue processing can be sequentially reversed by a carefully designed series of heating, dialysis, rehydration, and protein renaturation steps, carried out under appropriate solvation conditions. We have formulated this hypothesis on the basis of our strong preliminary data, which have shown that the reversal of formaldehyde-induced chemical changes to proteins is relatively facile in aqueous solutions but requires a different approach for tissue that has been dehydrated in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for high-throughput proteomic screening of FFPE tissues. This will improve practical interventions for the diagnosis, treatment, and prevention of cancer and will facilitate the development of therapeutic agents. Our studies are innovative because we have developed a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins that occur during histologic processing. At the completion of this project, it is our expectation to have established a comprehensive understanding of the protein modifications that occur during tissue histology, together with methods for optimally reversing these modifications. This knowledge should result in the ability to carry out proteomic analysis using FFPE tissue.