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.