Quantifying LpxC Activity by LC-MS

To quantify the activity of LpxC in vitro, the PaLpxC product and substrate were detected by LC–MS on an Agilent Technologies 1200 series HPLC system in line with an Agilent 6520 Q-TOF mass spectrometer with electrospray ionization–mass spectrometry operating in negative mode. Samples were separated on a Waters Symmetry C18 column (5 µm; 3.9 mm × 150 mm) with a matching column guard using the following method: flow rate = 0.5 ml min⁻¹, 95% solvent A (H₂O, 10 mM ammonium formate) and 5% solvent B (acetonitrile) for 5 min followed by a linear gradient of solvent B from 5–80% over 20 min. Agilent MassHunter Workstation Qualitative Analysis software version B.06.00 was used for analysing the MS data. The PaLpxC substrate (UDP-3-O-(R-3-hydroxydecanoyl)-N-acetylglucosamine) was quantified by monitoring the abundance of 776.21 m/z and resolved as a single peak, which was integrated to infer substrate concentration. The PaLpxC product (UDP-3-O-(R-3-hydroxydecanoyl)-glucosamine) was quantified by monitoring the abundance of 734.1944 m/z and often resolved as multiple peaks as reported previously^{43 (link)}, all of which were integrated to infer product concentration.
PaLpxC substrate and product from the aqueous fraction of methanol–chloroform-extracted whole-cell lysates were analysed by LC–MS/MS using the same settings as the LC–MS analysis described above with the following adaptations. Parent ions with m/z of 776.1986 (corresponding to the PaLpxC substrate) and m/z of 734.1872 (corresponding to the PaLpxC product) were targeted for MS/MS with a collision energy of 40. Fragment ions between 50 and 850 m/z were analysed. The relative abundance of the PaLpxC substrate and product were quantified by integrating the peaks observed for 776.1986 m/z and 734.1872 m/z, respectively. Raw LC–MS/MS data are available at 10.5281/zenodo.7455522 (ref. ⁴² ).