Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially 
valuable resource for retrospective biomarker discovery studies, and recent work 
indicates the feasibility of using shotgun proteomics to characterize FFPE 
tissue proteins. A critical question in the field is whether proteomes 
characterized in FFPE specimens are equivalent to proteomes in corresponding 
fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of 
frozen and FFPE specimens prepared from the same colon adenoma tissues. 
Following deparaffinization, rehydration, and tryptic digestion under mild 
conditions, FFPE specimens corresponding to 200 microg of protein yielded 
approximately 400 confident protein identifications in a one-dimensional reverse 
phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The 
major difference between frozen and FFPE proteomes was a decrease in the 
proportions of lysine C-terminal to arginine C-terminal peptides observed, but 
these differences had little effect on the proteins identified. No covalent 
peptide modifications attributable to formaldehyde chemistry were detected by 
analyses of the MS/MS datasets, which suggests that undetected, cross-linked 
peptides comprise the major class of modifications in FFPE tissues. Fixation of 
tissue for up to 2 days in neutral buffered formalin did not adversely impact 
protein identifications. Analysis of archival colon adenoma FFPE specimens 
indicated equivalent numbers of MS/MS spectral counts and protein group 
identifications from specimens stored for 1, 3, 5, and 10 years. Combination of 
peptide isoelectric focusing-based separation with reverse phase LC-MS/MS 
identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 
protein groups from FFPE tissue with at least two distinct peptide 
identifications per protein. Analysis of the combined frozen and FFPE data 
showed a 92% overlap in the protein groups identified. Comparison of gene 
ontology categories of identified proteins revealed no bias in protein 
identification based on subcellular localization. Although the status of 
posttranslational modifications was not examined in this study, archival samples 
displayed a modest increase in methionine oxidation, from approximately 17% 
after one year of storage to approximately 25% after 10 years. These data 
demonstrate the equivalence of proteome inventories obtained from FFPE and 
frozen tissue specimens and provide support for retrospective proteomic analysis 
of FFPE tissues for biomarker discovery.