Precision of Multiple Reaction Monitoring Mass Spectrometry Analysis of Formalin-Fixed, Paraffin-Embedded Tissue

We compared the reproducibility of multiple reaction
monitoring
(MRM) mass spectrometry-based peptide quantitation in tryptic digests
from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell
renal cell carcinoma tissues. The analyses targeted a candidate set
of 114 peptides previously identified in shotgun proteomic analyses,
of which 104 were detectable in FFPE and frozen tissue. Although signal
intensities for MRM of peptides from FFPE tissue were on average 66%
of those in frozen tissue, median coefficients of variation (CV) for
measurements in FFPE and frozen tissues were nearly identical (18–20%).
Measurements of lysine C-terminal peptides and arginine C-terminal
peptides from FFPE tissue were similarly reproducible (19.5% and 18.3%
median CV, respectively). We further evaluated the precision of MRM-based
quantitation by analysis of peptides from the Her2 receptor in FFPE
and frozen tissues from a Her2 overexpressing mouse xenograft model
of breast cancer and in human FFPE breast cancer specimens. We obtained
equivalent MRM measurements of HER2 receptor levels in FFPE and frozen
mouse xenografts derived from HER2-overexpressing BT474 cells and
HER2-negative Sum159 cells. MRM analyses of 5 HER2-positive and 5
HER-negative human FFPE breast tumors confirmed the results of immunohistochemical
analyses, thus demonstrating the feasibility of HER2 protein quantification
in FFPE tissue specimens. The data demonstrate that MRM analyses can
be performed with equal precision on FFPE and frozen tissues and that
lysine-containing peptides can be selected for quantitative comparisons,
despite the greater impact of formalin fixation on lysine residues.
The data further illustrate the feasibility of applying MRM to quantify
clinically important tissue biomarkers in FFPE specimens.