LC-MS/MS profiling of sample processing optimization 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. While 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 immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled with MRM-MS (targeting 515 analytes) to evaluate quantitatively the performance of three extraction protocols in combination with three trypsin digestion protocols (9 processes). An optimum process based on RapiGest buffer extraction and urea-based digestion was identified. The optimized process is generally amenable and enables highly reproducible, multiplex, standardizable quantitative MRM in archival specimens.