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

• Published in: Journal of proteome research Vol. 15; no. 8; pp. 2717 - 2728
• Main Authors: Kennedy, Jacob J, Whiteaker, Jeffrey R, Schoenherr, Regine M, Yan, Ping, Allison, Kimberly, Shipley, Melissa, Lerch, Melissa, Hoofnagle, Andrew N, Baird, Geoffrey Stuart, Paulovich, Amanda G
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 05-08-2016
• Subjects: Biomarkers - analysis; Cells - chemistry; Formaldehyde; Humans; Isotope Labeling; Mass Spectrometry - methods; Paraffin Embedding; Proteomics - methods; Tissue Fixation; Tissue Preservation - methods; archived tissue; immunoaffinity enrichment; multiple reaction monitoring; peptide assays; FFPE; targeted proteomics; mass spectrometry
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.6b00245

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 (R 2 = 0.94) and immuno-MRM (R 2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.