Binding of small molecules to macromolecules such as proteins has a deep physiological impact as it alters the physico-chemical properties and the functionality of the protein including alteration of enzymatic parameters through allosteric regulation and change in protein-protein interactions due to substantial conformational changes upon small molecule binding . There are a variety of directed and unbiased experimental approaches to determine binding of small molecules to proteins and to assess their binding affinity . Traditional biochemical approaches require purified protein and are not highthroughput. One of the most successful experimental approaches suitable for screening of a large number of candidate receptors for binding with candidate small ligand is the differential radial capillary action of ligand assay (DRaCALA), which does not require purification of the expressed protein, but uses whole bacterial cell lysates containing plasmid-expressed candidate gene products . Thereby, small ligand binding to a protein is assessed by the spot intensity and diameter upon spotting the protein extract containing ligand onto a protein-binding nitrocellulose membrane. Binding of the radioactive ligand to the macromolecule creates a small intensive spot reflecting the protein location, while, upon non-binding, the ligand signal is more extended, but overall equally intensive reflecting the free ligand distribution over a larger spot area created by capillary forces. Cyclic di-nucleotides are ubiquitous second messengers in Bacteria, however, the abundance of cyclic di-GMP turnover proteins is still not reflected by the number of identified receptors . DRaCALA has been frequently used to identify receptors for cyclic di-nucleotides, in particular cyclic di-GMP and cyclic di-AMP, which are ubiquitous second messengers in Gram-negative and Gram-positive Bacteria . Thereby lysates of bacterial libraries containing all plasmid-expressed gene products of an organism have been screened for cyclic di-nucleotide binding proteins. With this experimental approach, receptors involved in, for example, regulation of cellulose biosynthesis such as BcsE and maintenance of osmohomeostasis and potassium transport have been identified . A radioactive ligand, as in the originally described approach, needs to be prepared and labeling is not necessarily readily applicable to every small molecular compound. However, MALDI-TOF MS or imaging mass spectrometry can be used to detect binding of a ligand to a protein receptor on the nitrocellulose membrane. Thereby, assessment of binding can be achieved by measuring the ligand concentration at the application spot and at the edge of the larger spot area that the liquid reached by capillary action (Fig. ). To perform this assay, a single colony of the E. coli strains (in case of the control with the vector containing a cloned cyclic di-GMP binding protein (YcgR or BcsE) and its relevant non-binding mutant) were inoculated 1:100 from an overnight culture in LB medium with the relevant antibiotic. After induction with 0.1% L-arabinose at 30°C for four hours, cells were harvested by resuspension in binding buffer (10 mM Tris pH 8.0, 100 mM NaCl, 5 mM MgCl2, in Chromasolv H2O; with 100 µl lysozyme (500 µg/ml) per OD600=3 ( ). Cells were disrupted by two freeze-thaw cycles of 1h at room temperature and at -80°C. 18 µl cell lysate and 2 µl 10 µM c-di-GMP were mixed, incubated 10 min at room temperature and 2 µl were spotted on a nitrocellulose membrane (Schleicher & Schuell Protan TM , BA85). After the membrane had completely dryed, equally sized membrane pieces at the application spot and the edge of the diffused liquid were cut out with a scalpel. 2 µl 5 mg/ml alpha-cyano-4-hydroxy-cinnamic acid matrix in 50% acetonitrile and 0.05% triflouroacetic acid were applied on a MALDI MassTech 96 spot sample plate, followed by the membrane piece and again 2 µl matrix. The matrix was left to dry at room temperature, the membrane removed and the sample measured in a QExactive, Thermo Scientific, with a MassTech AP MALDI PDF Ion source. Settings of the MALDI source were sheath gas: 0; aux gas: 0; sweep gas: 0; spray voltage: 3,75kV; spray current [µA]; capillary temperature: 280; S-lens RF level: 100.0; 5 min acquisition time, 2 microscans; lock masses OFF; 1e 5 AGC target; maximal inject time 1.000. Signals for cyclic di-GMP were observed as expected at around 691.1 [M+H] + and 713.1 [M+Na] + . In order to validate the procedure, we first expressed two previously characterized cyclic di-GMP binding proteins and their respective binding mutants, namely the cellulose biosynthesis enhancer BcsE and its non-binding mutant BcsER415D and the flagella motor 'backstop-break' protein YcgR and its nonbinding mutant YcgRR118D in E. coli Top10 . Measurement of the cyclic di-GMP counts from cell lysates expressing these receptors after incubation with cyclic di-GMP showed higher counts derived from the membrane taken from the application spot than from the periphery. In contrast, counts were similar when membranes from cell lysates expressing the non-binding protein variants were investigated after incubation with cyclic di-GMP (Fig. ). YciR, an enzyme with opposing diguanylate cyclase and phosphodiesterase activities, has been initially characterized to downregulate expression of the agar-grown rdar biofilm morphotype in Escherichia coli and Salmonella typhimurium by a mechanism independent of its catalytic activities involving cyclic di-GMP sensing and proteinprotein interactions . Natural YciR protein variants encoded by semiconstitutive rdar biofilm strains, though, failed to efficiently downregulate this morphotype, even when overexpressed. Sensing and binding of cyclic di-GMP might be a determinative factor for the different variant behavior. To assess whether two YciR protein variants differentially bind cyclic di-GMP and to assess their binding capacity, we choose to express the two variants of the opposing activity enzyme YciR, YciRFec101, that has been previously shown to downregulate the rdar morphotype, and YciRTOB1 that is nearly invariant to rdar morphotype expression upon plasmid-based expression . Upon expression of YciR from the commensal E. coli strain Fec101, the MALDI-TOF MS -based DRaCALA binding assay showed higher counts for the membrane derived from the application spot compared to the membrane from the periphery suggesting binding of cyclic di-GMP to YciRFec101. In contrast, membranes from the application spot and the periphery of the membrane for the YciRFec101-K581A mutant and a YciR double mutant in the GGDEF and EAL motif, YciRFec101-D316A,E317A/E440A, abolishing the two catalytic activities of the combined diguanylate cyclase and phosphodiesterase, showed similar counts suggesting those protein variants are either non-binding or not being expressed under the respective assay conditions. Equally, the counts from the application spot and the membrane periphery for YciRTOB1 were even higher for the periphery suggesting that YciRTOB1 does not bind cyclic di-GMP (Fig. ).