Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as 
an alternative to fresh/frozen tissue for retrospective protein biomarker 
discovery. However, during the fixation process, proteins undergo degradation 
and cross-linking, making conventional protein analysis technologies 
problematic. In this study, we have compared several extraction and separation 
methods for the analysis of proteins in FFPE tissues. Incubation of tissue 
sections at high temperature with a novel extraction buffer (20 mM Tris-HCl, pH 
8.8, 2% SDS, 1% beta-octylglucoside, 200 mM DTT, 200 mM glycine, and a mixture 
of protease inhibitors) resulted in improved protein recovery. Protein 
separation by 1-DE followed by LC-ESI MS/MS analysis was the most effective 
approach to identify proteins, based on the number of peptides reliably 
identified. Interestingly, a number of peptides were identified in regions of 
the 1DE not corresponding to their native molecular weights. This is an 
indication of the formation of protein-protein complexes by cross-linking, and 
of protein fragmentation due to prolonged sample storage. This study will 
facilitate the development of future proteomic analysis of FFPE tissue and 
provide a tool for the validation in archival samples of biomarkers of exposure, 
prognosis and disease.