N-Myristoyl-d-asparagine (colibactin prodrug motif) quantification by liquid chromatography-mass spectrometry.

The colibactin prodrug motif was quantified as previously described (36). Briefly, precultivated strains were grown in DMEM-HEPES at 37°C for 18 h under shaking (240 rpm). Supernatants of cultures were obtained by centrifugation of bacterial cells at 3,200 × g for 15 min and were filtered on 0.2-μm-pore membranes. Each strain was cultured in triplicate (derived from three independent clones), and each supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Quantification experiments were performed with ultraperformance liquid chromatography high-resolution/heated electrospray ionization mass spectrometry (UPLC-HR/HESI-MS). The data were recorded on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer coupled to a Dionex Ultimate 3000 UPLC. The following solvent gradient (A = H₂O + 0.1% formic acid, B = acetonitrile + 0.1% formic acid with B at 30% from0 to 1 min, 30 to 95% from 1 to 6 min, and 95% from 6 to 7 min at a flow rate of 0.5 ml/min) was used on a Phenomenex Kinetex 5-μm EVO C₁₈ (50- by 2.1-mm) column at 30°C. The mass spectrometer was operated in positive-ionization mode at a scan range of 200 to 500 m/z and a resolution of 35,000. The spray voltage was set to 3.5 kV, the S-lens to 35, the auxiliary gas heater temperature to 438°C, and the capillary temperature to 270°C. Absolute quantification was achieved by using a Schotten-Baumann reaction-derived N-myristoyl-l-asparagine (isomer of the N-myristoyl-d-asparagine colibactin cleavage product) as a standard. Data were obtained from undiluted cell-free sample supernatants and analyzed for N-myristoyl-d-asparagine, and concentrations were calculated using Thermo Xcalibur 2.2 Quan Browser.