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Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues
- Source :
- Journal of Proteome Research. 15:2717-2728
- Publication Year :
- 2016
- Publisher :
- American Chemical Society (ACS), 2016.
-
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.
- Subjects :
- Proteomics
0301 basic medicine
Analyte
Tissue Fixation
Formalin fixed paraffin embedded
Cells
Biochemistry
Article
Mass Spectrometry
03 medical and health sciences
Formaldehyde
Humans
Multiplex
Bradford protein assay
Protocol (science)
Paraffin Embedding
Chromatography
integumentary system
Chemistry
Selected reaction monitoring
General Chemistry
Molecular biology
030104 developmental biology
Isotope Labeling
Tissue Preservation
Trypsin Digestion
Biomarkers
Subjects
Details
- ISSN :
- 15353907 and 15353893
- Volume :
- 15
- Database :
- OpenAIRE
- Journal :
- Journal of Proteome Research
- Accession number :
- edsair.doi.dedup.....bcc71523047dc4e35499b12c770b04f5