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IsoTopics™ – April 2016

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

With the need for highly robust quantitative assays for phospho-signaling in mind, Paulovich, A. et al. developed an antibody-free, multiplex IMAC-MRM method with isotopically labeled peptide standards to quantify 107 phosphosites (93 proteins) in human cell lysates. These phosphopeptides are involved in DNA damage-response (DDR) signaling pathways and were quantified with excellent linearity (≥3 orders of magnitude) and a high degree of precision (<13% intra- and inter-assay CV). Opportunities to assess and expand this phosphopeptide panel are available for cell signaling quantitation through access to the online resources provided with this article.


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.

Kennedy JJ, Yan P, Zhao L, Ivey RG, et al.

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