Overlap of Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Matched Tissues for Proteomics and Phosphoproteomics

Many liquid chromatography-mass spectrometry (LC-MS) studies have compared formalin-fixed paraffin-embedded (FFPE) tissues with matched fresh-frozen (FF) tissues to examine the effect of preservation techniques on the proteome; however, few studies have included the phosphoproteome. A high degree of overlap and correlation between the two preservation techniques would demonstrate the importance of FFPE tissues as a valuable biomedical resource. Our aim was to quantitatively compare the proteome and phosphoproteome of matched FFPE and FF tissues using data-independent acquisition LC-MS. Four organs from three rats were cut in half to produce matched FFPE and FF tissue pairs. Excellent overlaps of 85-97% for the proteome and 82-98% for the phosphoproteome were observed, depending on the organ type, between the two preservation techniques. Most of the unique identifications were found in FF with less than 0.3% being unique to FFPE tissues. Strong agreement between FFPE and FF matched tissue pairs was observed with Pearson correlation coefficients of 0.93-0.97 and 0.79-0.87 for the proteome and phosphoproteome, respectively. Digestion efficiency was slightly higher in FFPE (92-94%) than in FF tissues (86-89%), and a search of a data subset for formaldehyde induced chemical modifications revealed that only 0.05% of precursors were unique to FFPE tissues. This suggests that with quality sample preparation methods it is not necessary to include formaldehyde induced chemical modifications when analyzing FFPE tissues. We attribute the lower number of identifications in FFPE tissues to inaccurate peptide quantitation, which resulted in a lower MS peptide load and tryptic peptide enrichment load. Our results demonstrate that both proteomic and phosphoproteomic analyses of FFPE and FF tissues are highly comparable and highlight the suitability of FFPE tissues for both proteomic and phosphoproteomic analysis.