title: Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues.
abstract: 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.
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States.
authors.forename: Jacob J
authors.initials: JJ
authors.lastname: Kennedy
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Whiteaker
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States.
authors.forename: Regine M
authors.initials: RM
authors.lastname: Schoenherr
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States.
authors.forename: Ping
authors.initials: P
authors.lastname: Yan
authors.affiliation: Department of Pathology, Stanford University , Stanford, California 94305 United States.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Allison
authors.affiliation: Department of Laboratory Medicine, University of Washington , Seattle, Washington 98195 United States.
authors.forename: Melissa
authors.initials: M
authors.lastname: Shipley
authors.affiliation: Department of Laboratory Medicine, University of Washington , Seattle, Washington 98195 United States.
authors.forename: Melissa
authors.initials: M
authors.lastname: Lerch
authors.affiliation: Department of Laboratory Medicine, University of Washington , Seattle, Washington 98195 United States.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Hoofnagle
authors.affiliation: Department of Laboratory Medicine, University of Washington , Seattle, Washington 98195 United States.
authors.forename: Geoffrey Stuart
authors.initials: GS
authors.lastname: Baird
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States.
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Paulovich
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002477:Cells / Q000737:chemistry*; D005557:Formaldehyde; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods; D016612:Paraffin Embedding; D040901:Proteomics / Q000379:methods*; D016707:Tissue Fixation; D014021:Tissue Preservation / Q000379:methods
chemical_list: D015415:Biomarkers; D005557:Formaldehyde
keywords: FFPE; archived tissue; immunoaffinity enrichment; mass spectrometry; multiple reaction monitoring; peptide assays; targeted proteomics