Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling.

Molecular & cellular proteomics : MCP

PubMedID: 25987412

Whiteaker JR, Zhao L, Yan P, Ivey RG, Voytovich UJ, Moore HD, Lin C, Paulovich AG. Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling. Mol Cell Proteomics. 2015;.
In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show 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 interassay 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 shows the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and nonmodified peptides, showing feasibility for development of targeted assay panels to cell signaling networks.