title: Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling.
abstract: 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.
authors.affiliation: From the ‡Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109.
authors.forename: Jeffrey R
authors.identifier: http://orcid.org/0000-0001-5042-8580
authors.initials: JR
authors.lastname: Whiteaker
authors.affiliation: From the ‡Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.affiliation: From the ‡Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109.
authors.forename: Ping
authors.initials: P
authors.lastname: Yan
authors.affiliation: From the ‡Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Ivey
authors.affiliation: From the ‡Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109.
authors.forename: Uliana J
authors.initials: UJ
authors.lastname: Voytovich
authors.affiliation: From the ‡Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109.
authors.forename: Heather D
authors.initials: HD
authors.lastname: Moore
authors.affiliation: From the ‡Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109.
authors.forename: Chenwei
authors.initials: C
authors.lastname: Lin
authors.affiliation: From the ‡Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109 apaulovi@fredhutch.org.
authors.forename: Amanda G
authors.identifier: http://orcid.org/0000-0001-6532-6499
authors.initials: AG
authors.lastname: Paulovich
mesh_terms: D000328:Adult; D001681:Biological Assay; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D014644:Genetic Variation / Q000187:drug effects; D006801:Humans; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods*; D009369:Neoplasms / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D007202:Indicators and Reagents; D010748:Phosphopeptides; D047428:Protein Kinase Inhibitors