In conclusion, a MALDI-TOF MS-based DRaCALA assay to identify cyclic dinucleotide binding proteins was successfully implemented which should save experiment time, as production of radioactive cyclic di-GMP is not necessary. Future miniaturization of the experiment will save material and optimization of the choice of the protein-binding matrix with optimal capillary forces for small compound diffusion will optimize the detection process. Furthermore, the approach can be transferred to measure binding of other small ligands and molecules where methodological restrictions do not allow read radioactive labelling. Ratio of counts derived from representative absolute values measured by FTMS MALDI-TOF on the membrane at the application spot and the peripheral membrane (range m/z 691.0950 -691.1050). A value >1 is indicative for cyclic di-GMP binding to the expressed protein (black bars), a value <1 indicates no binding (gray bars). A Proof of principle shown with the known cyclic di-GMP receptors BcsE and YcgR and their respective non-binding catalytic mutants . B Cyclic di-GMP binding capabilities of the cyclic di-GMP metabolizing protein YciR from the commensal E. coli strains TOB1 and Fec101. While YciRFec101 binds c-di-GMP, the respective catalytic mutants and YciRTOB1 do not. dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virulence, Proc Natl Acad Sci U S A, 114 (2017) 13006-13011.