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

描述（由申请人提供）：癌症是美国发病率和过早死亡的主要原因; 2005年，癌症导致超过550，000人死亡。高通量基因组学和蛋白质组学方法为发展癌症分子特征的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无法及时与疾病的临床病程直接相关。世界范围内的组织库包含数百万福尔马林固定、石蜡包埋（FFPE）的标本，这些标本可以通过提供大量的组织来克服这一限制，其中已经建立了疾病和治疗的临床过程。然而，通过高通量蛋白质组学方法分析存档FFPE组织受到福尔马林固定的不利影响的阻碍。我们最近证明，当溶菌酶组织替代物（FFPE组织的模型）在65-80 oC的高静水压力（45，000 psi）下在含有2%SDS的Tris缓冲液（pH 4）中处理时，甲醛诱导的蛋白加合物和交联几乎定量逆转。我们还通过MS鉴定了在高压下提取的5-蛋白FFPE组织替代物中的所有蛋白质，其具有优异的序列覆盖率，与相同的天然蛋白质混合物相当。这些结果表明，提高静水压力是一种有前途的方法，从FFPE组织中回收蛋白质的形式适合于蛋白质组学分析，因此是值得进一步研究。本提案的目的是在中等温度（=65 ℃）下使用升高的静水压力，以开发一种显著改进的方法，用于从FFPE组织中回收蛋白质用于蛋白质组学分析。在目标1中，我们将使用升高的压力来优化从复杂组织替代物中定量提取蛋白质的方法，并逆转其甲酰胺诱导的修饰。在目标2中，我们将使用目标1中开发的方法进行一系列蛋白质组学分析，将新鲜细胞裂解物中鉴定的蛋白质与来自处理为FFPE琼脂糖细胞塞的相同细胞的蛋白质进行比较。在目标3中，我们将确认优化的压力提取方法可用于对真实的FFPE组织进行蛋白质组学分析。使用高静水压并辅以适度加热（=65 oC）是一种创新且高效的方法，可从FFPE组织中提取完整蛋白质并逆转其由甲醛诱导的修饰。这些研究的基本原理是，它们的成功完成将最终导致改善癌症诊断，治疗和预防的实际干预措施，并促进治疗药物的开发。

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.