Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues
Kennedy JJ, Whiteaker JR, Schoenherr RM, Yan P, Allison K, Shipley M, Lerch M, Hoofnagle AN, Baird GS, Paulovich AG1
- Organism: Human
- Instrument: Sciex 5500 QTRAP
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 512 analytes) to evaluate quantitatively the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). An optimum process based on RapiGest buffer extraction and urea-based digestion was identified. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 246 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.
Created on 3/18/16, 1:49 PM