Conical 96 well plates

Synthesis of radiolabeled c-di-GMP using YdeH and UV light-induced cross-linking experiments in conical 96-well plates (Greiner Bio-One) were performed as described recently (Steiner et al., 2013 (link); Christen et al., 2005 (link)). Briefly, 2 μM protein was incubated for 10 min at room temperature with c-[³³P]-di-GMP in a total volume of 20 μl using PBS as buffer. As a control BSA was included. For competition experiments unlabeled nucleotides were incubated with the protein for 15 min prior the addition of c-[³³P] -di-GMP. The 96-well plates were then UV-irradiated at 254 nm for 20 min using a Bio-Link crosslinker (Vilber Lourmat, France). After addition of 5 μl loading dye and boiling for 5 min the samples were subjected to SDS-PAGE. Proteins were analyzed by Coomassie staining and autoradiography. Band intensities were quantified using the ImageJ64 software and the data were fitted using GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego California USA). The K_d was calculated by non-linear regression using the ‘One site – Total binding’ equation.

Pooled and dried samples were resuspended in 20 μl 2% (vol/vol) acetonitrile for fractionation. Basic reversed phase fractionation was performed on a quaternary Agilent 1290 Infinity II UPLC system equipped with a Kinetex Evo-C₁₈ column (150 by 2.1 mm, 2.6 μm, 100 Å, Phenomenex) that was operated at 40°C. Solvent A consisted of high-performance liquid chromatography (HPLC)-grade H₂O, solvent B consisted of 100% acetonitrile, and solvent C consisted of 25 mM ammonium bicarbonate. Fractionation was carried out at a constant flow rate of 100 μl/minute using a linear gradient from 2 to 25% (vol/vol) acetonitrile within 50 min, followed by column washing and equilibration. Over the whole gradient, solvent C was kept constant at 10% (vol/vol). In total, 32 fractions with 200 μl per fraction were collected in conical 96-well plates (Greiner). The organic solvent was removed in a vacuum concentrator (default settings for organic solvents) for 1 h and fractions were concatenated into 8 final samples as described before (64 (link)). Peptides were acidified with formic acid (Thermo Fisher Scientific) to a final concentration of 1% (vol/vol), desalted using OASIS HLB 96-well cartridges (Waters), dried down at room temperature, and resuspended in 30 μl 2% (vol/vol) acetonitrile, 0.1% (vol/vol) trifluoroacetic acid (TFA) prior to mass spectrometry (MS) analysis.