Synthetic phosphopeptides: From spike-in standards to affinity tools for protein-protein interaction studies

Abstract Synthetic isotope labeled phosphopeptidesare valuable tools for the quantification and validation of phosphoproteomedata. Here, we report that the same set of phosphopeptides, which are used as spike-in standards, can be successfully applied for identification of stimulus specific protein-protein interactionsmediated by the respective phosphorylation sites. As a proof-of- concept, binding of two γ H2AX (pS139) phosphosite specific interaction partners, MDC1and 53BP1, was confirmed and elevated binding affinity was revealed in response to ionizing radiation. Our strategy is generally applicable and enables multiplexed validation and functional analysisof phosphorylation sites offering great potential for the follow-up of phosphoproteomestudies.