Recovery of Proteins from Formalin-fixed Tissues under High Hydrostatic Pressure

描述(申请人提供)：癌症是美国发病率和过早死亡的主要原因；2005年，它导致了550,000多人死亡。高通量的基因组和蛋白质组学方法为发展癌症的分子特征知识带来了巨大的希望，这些知识可以转化为诊断、治疗和预防这种疾病的实际干预措施。不幸的是，使用新鲜或冷冻组织的蛋白质组学研究不能及时与临床病程直接相关。世界各地的组织储存库包含数百万福尔马林固定的石蜡包埋(FFPE)标本，这些标本可以通过提供大量已建立临床病程和治疗的组织来克服这一限制。然而，用高通量蛋白质组学方法分析档案FFPE组织受到福尔马林固定的不利影响。我们最近证明，当溶菌酶组织替代物，一个FFPE组织的模型，在65-80 oC的高静水压力(45000 Psi)下，在含有2%SDS的Tris缓冲液中处理时，甲醛诱导的蛋白质加合物和交联链几乎被定量逆转。我们还用MS鉴定了高压提取的5蛋白FFPE组织替代物中的所有蛋白质，具有极好的序列覆盖率，可与相同的天然蛋白质混合物相媲美。这些结果表明，高静水压是一种从FFPE组织中回收适合蛋白质组分析的蛋白质的有前景的方法，因此值得进一步研究。这项建议的目的是利用在中等温度(=65oC)下提高静水压力来开发一种显著改进的从FFPE组织中回收蛋白质的方法，用于蛋白质组学分析。在目标1中，我们将利用加压来优化一种从复杂组织替代物中定量提取蛋白质并逆转其甲醛诱导的修饰的方法。在目标2中，我们将使用目标1中开发的方法来进行一系列蛋白质组学分析，比较在新鲜细胞裂解物中鉴定的蛋白质与作为FFPE琼脂糖凝胶细胞塞加工的相同细胞中的蛋白质。在目标3中，我们将确认优化的压力提取方法可以用于对真实的FFPE组织进行蛋白质组学分析。利用高静水压和中等温度(=65oC)是一种从FFPE组织中提取全蛋白并逆转其甲醛诱导的修饰的创新和高效的方法。这些研究的基本原理是，它们的成功完成最终将导致改善癌症诊断、治疗和预防的实际干预措施，并促进治疗剂的开发。

DESCRIPTION (provided by applicant): Cancer is a leading cause of morbidity and premature mortality in the United States; it led to over 550,000 deaths in 2005. High-throughput genomic and proteomic methods hold great promise for developing knowledge of the molecular characteristics of cancer, which can be translated into practical interventions for the diagnosis, treatment, and prevention of this disease. Unfortunately, proteomic studies using fresh or frozen tissues cannot be related directly to the clinical course of disease in a timely way. Tissue repositories world-wide contain millions of formalin-fixed, paraffin-embedded (FFPE) specimens that could overcome this limitation by providing large numbers of tissues for which the clinical course of disease and treatment have been established. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hampered by the adverse effects of formalin fixation. We recently demonstrated nearly quantitative reversal of formaldehyde-induced protein adducts and cross-links when lysozyme tissue surrogates, a model for FFPE tissues, were processed under high hydrostatic pressure (45,000 psi) at 65-80 oC in Tris buffer, pH 4, containing 2% SDS. We have also identified by MS all of the proteins in a 5-protein FFPE tissue surrogate extracted under high pressure with excellent sequence coverage, comparable to an identical native protein mixture. These results indicate that elevated hydrostatic pressure is a promising approach for recovering proteins from FFPE tissues in a form suitable for proteomic analysis, and thus is worthy of further study. The objective of this proposal is to use elevated hydrostatic pressure at moderate temperatures (=65 oC) to develop a significantly improved method for recovering proteins from FFPE tissues for proteomic analysis. In Aim 1, we will use elevated pressure to optimize a method for the quantitative extraction of proteins from complex tissue surrogates and the reversal of their formaldehyde-induced modifications. In Aim 2 we will use methods developed in Aim 1 to conduct a series of proteomic analyses comparing proteins identified in fresh cell lysates with those from the same cells processed as FFPE agarose-cell plugs. In Aim 3, we will confirm that the optimized pressure extraction methods can be used to perform proteomic analyses with real FFPE tissue. The use of high hydrostatic pressure supplemented with moderate heating (=65 oC)is an innovative and highly effective approach for the extraction of whole proteins from FFPE tissues and the reversal of their formaldehyde-induced modifications. The rationale for these studies is that their successful completion will ultimately lead to improved practical interventions for the diagnosis, treatment, and prevention of cancer and facilitate the development of therapeutic agents.