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

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• 作者：Jacob J. Kennedy ; Jeffrey R. Whiteaker ; Regine M. Schoenherr ; Ping Yan ; Kimberly Allison ; Melissa Shipley ; Melissa Lerch ; Andrew N. Hoofnagle ; Geoffrey Stuart Baird ; Amanda G. Paulovich
• 刊名：Journal of Proteome Research
• 出版年：2016
• 出版时间：August 5, 2016
• 年：2016
• 卷：15
• 期：8
• 页码：2717-2728
• 全文大小：526K
• 年卷期：0
• ISSN：1535-3907

文摘

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 (R² = 0.94) and immuno-MRM (R² = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.