Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues

Despite
a clinical, economic, and regulatory imperative to develop
companion diagnostics, precious few new biomarkers have been successfully
translated into clinical use, due in part to inadequate protein assay
technologies to support large-scale testing of hundreds of candidate
biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although
the feasibility of using targeted, multiple reaction monitoring mass
spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has
been demonstrated, protocols have not been systematically optimized
for robust quantification across a large number of analytes, nor has
the performance of peptide immuno-MRM been evaluated. To address this
gap, we used a test battery approach coupled to MRM-MS with the addition
of stable isotope-labeled standard peptides (targeting 512 analytes)
to quantitatively evaluate the performance of three extraction protocols
in combination with three trypsin digestion protocols (i.e., nine
processes). A process based on RapiGest buffer extraction and urea-based
digestion was identified to enable similar quantitation results from
FFPE and frozen tissues. Using the optimized protocols for MRM-based
analysis of FFPE tissues, median precision was 11.4% (across 249 analytes).
There was excellent correlation between measurements made on matched
FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex,
standardizable, quantitative MRM in archival tissue specimens.