Synergi 4μ hydro rp 80a column

C-di-GMP was extracted from S. Typhimurium grown in 96-well plates using a method adapted from (Hickman and Harwood, 2008 (link); Irie et al., 2012 (link)). Briefly, all experiments were performed on independent cultures in biological triplicates. C-di-GMP was extracted by addition of 9 µl of 70% perchloric acid to each well of the 96 well plate and incubation on ice for 1 hour. After an hour, attached cells were dislodged by pipetting and the supernatant was collected and neutralized using potassium bicarbonate. 2-chloro AMP was used as an internal standard. Liquid chromatography MS/MS measurements were performed using an Acuity UPLC with a Synergi 4μ Hydro RP 80A column and a C18 Guard Cartridge (Phenomenex) on a Premier XL triple-quadrupole electrospray mass spectrometer (Waters). The m/z 691 > 152 transition was used for c-di-GMP and 382 > 170 for 2-chloro-AMP. The cone voltages and collision energies were 40 V/30 eV and 35 V/20 eV, respectively. Thirty microliters of each sample was injected and the ratio of area under the curve of the c-di-GMP channel signal (retention time= 1.6 minutes) was divided by the area under the curve of the 2-chloroAMP signal (retention time= 2.1 minutes). A standard curve of 0 nM to 100 nM c-di-GMP containing 2-chloroAMP was used to quantify c-di-GMP for all samples. C-di-GMP concentration was normalized to total protein concentration.