Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue.

Autor: Daniel V T Catenacci, Wei-Li Liao, Sheeno Thyparambil, Les Henderson, Peng Xu, Lei Zhao, Brittany Rambo, John Hart, Shu-Yuan Xiao, Kathleen Bengali, Jamar Uzzell, Marlene Darfler, David B Krizman, Fabiola Cecchi, Donald P Bottaro, Theodore Karrison, Timothy D Veenstra, Todd Hembrough, Jon Burrows
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Zdroj: PLoS ONE, Vol 9, Iss 7, p e100586 (2014)
Druh dokumentu: article
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0100586
Popis: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for 'high Met' expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue.After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH).Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (
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