title: Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.
abstract: A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.
authors.affiliation: From the ‡ Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109;
authors.forename: Jacob J
authors.initials: JJ
authors.lastname: Kennedy
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: 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: 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;
authors.forename: Era L
authors.initials: EL
authors.lastname: Pogosova-Agadjanyan
authors.affiliation: From the ‡ Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109;
authors.forename: Derek L
authors.initials: DL
authors.lastname: Stirewalt
authors.affiliation: §University of Washington, 1959 NE Pacific St., Seattle, Washington 98195.
authors.forename: Kerryn W
authors.initials: KW
authors.lastname: Reding
authors.affiliation: From the ‡ Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109;
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Whiteaker
authors.affiliation: From the ‡ Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, Washington 98109; apaulovi@fredhutch.org.
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Paulovich
mesh_terms: D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004249:DNA Damage / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008670:Metals / Q000737:chemistry; D010748:Phosphopeptides / Q000096:biosynthesis* / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D040901:Proteomics; D015398:Signal Transduction / Q000235:genetics
chemical_list: D008670:Metals; D010748:Phosphopeptides