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

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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.