Identification of Covalent Modifications Regulating Immune Signaling Complex Composition and Phenotype

ABSTRACT Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post-transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here we devised M odifications, I nteractions and P henotypes by A ffinity P urification M ass S pectrometry (MIP-APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS-based quantification. We report the time-resolved interplay of more than 50 previously undescribed modification and hundreds of protein-protein interactions of 19 immune protein complexes in monocytes. Validation of interdependecies between covalent, reversible and functional protein complex regulations by knock-out or site-specific mutation, revealed isgylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll-like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p-38 (MAPK14). Our method provides a fast and cost-effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells. Highlights Experimental framework to reveal dynamic signaling checkpoints in primary cells Identification of crosstalk between protein modifications and interactions in signaling complexes Discovery of TRAF2 isgylation, phosphorylation and ARHGEF18 interaction in monocytes Differential drug mode of action for p-38 (MAPK14) kinase inhibitors Graphical abstract