Peptide immunoaffinity enrichment and targeted mass spectrometry enables multiplex, quantitative pharmacodynamic studies of phospho-signaling.

Publication Type:

Journal Article


Molecular & cellular proteomics : MCP (2015)


In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semi-quantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We demonstrate the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra- and inter-assay repeatability. The linear range was ≥3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median inter-assay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure-response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic (kinase) inhibitor. The study demonstrates the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and non-modified peptides, demonstrating feasibility for development of targeted assay panels to cell signaling networks.