Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phosphosignaling

Kennedy, J.J.; Yan, P.; Zhao, L.; et al.

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.