Journal
MOLECULAR & CELLULAR PROTEOMICS
Volume 14, Issue 8, Pages 2261-2273Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.O115.050351
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Funding
- National Cancer Institute of the National Institutes of Health (NIH) Clinical Proteomics Tumor Analysis Consortium Initiative [U24CA160034]
- Specialized Programs of Research Excellence (SPORE) [P50CA138293]
- Paul G. Allen Family Foundation (PGAFF)
- NATIONAL CANCER INSTITUTE [U24CA160034, P50CA138293] Funding Source: NIH RePORTER
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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.
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