An experimental strategy for the identification of AMP ylation targets from complex protein samples

AMP ylation is a posttranslational modification (PTM) that has recently caught much attention in the context of bacterial infections as pathogens were shown to secrete F ic proteins that AMP ylate R ho GTP ases and thus interfere with host cell signaling processes. Although F ic proteins are widespread and found in all kingdoms of life, only a small number of AMP ylation targets are known to date. A major obstacle to target identification is the limited availability of generic strategies allowing sensitive and robust identification of AMP ylation events. Here, we present an unbiased MS‐based approach utilizing stable isotope‐labeled ATP . The ATP isotopes are transferred onto target proteins in crude cell lysates by in vitro AMP ylation introducing specific reporter ion clusters that allow detection of AMP ylated peptides in complex biological samples by MS analysis. Applying this strategy on the secreted F ic protein B ep2 of B artonella rochalimae , we identified the filamenting protein vimentin as an AMP ylation target that was confirmed by independent assays. Vimentin represents a new class of target proteins and its identification emphasizes our method as a valuable tool to systematically uncover AMP ylation targets. Furthermore, the approach can be generically adapted to study targets of other PTM s that allow incorporation of isotopically labeled substrates.