Agilent 6130 single quadrupole mass spectrometer

To examine the activity of PKAL-1 and the PKAL-1(K488A) mutant, an LC–MS-based assay was used⁴⁹ . A 50 µL reaction mixture contained 100 mM potassium phosphate at pH 7.0, 5 mM MgCl₂, 5 mM CoA, 5 mM ATP, 0.1% Triton X-100, and 1 mM fatty acid substrate. The reaction was initiated by adding 2 µL of 2 mg/mL purified PKAL-1 or PKAL-1(K488A) enzyme at 25 °C for 2 h. 50 µL methanol was added to quench the reaction, and the reaction was vortexed and centrifuged. A 5 µL supernatant was used for LC–MS analysis on an Agilent 6130 single quadrupole mass spectrometer in both positive and negative full-scan modes, mass range 150–1500, 125 V fragmentor voltage, 0.15 min peak width, and 2.20 s cycle length. Mobile phase A was water with 10 mM ammonium acetate, and mobile phase B was acetonitrile. The LC gradient was started from 95% A for 2 min and then ramped up to 100% B over 24 min.

Purified ACS-7, ACS-7(E339A), and ACS-7(E339A,S186A,S187A) were concentrated to 2 mg/mL. 2 μL of the 2 mg/mL enzyme was used in each 50 μL reaction mixture, which gave a 1.57 μM final concentration. The reaction mixture contained 100 mM KPO₄ pH 7.0, 5 mM ATP, 5 mM MgCl₂, 5 mM CoA, and 100 μM IC-asc-C9 or ICA as substrates. Reaction mixtures were incubated at 25°C for 10 min, 1 hr, or 2 hr. 50 μL of methanol was added to the reaction mixtures to quench them, and 5 μL of the 100 μL 1:1 reaction/methanol mixture was analyzed by LC-MS directly. LC-MS analysis was performed with an Agilent 6130 single quadrupole mass spectrometer, operating in both positive and negative modes, using a method adapted from a previously published one (Zhang et al., 2013 (link)). The LC conditions were holding for 2 min at 95% solvent A (water with 10 mM ammonium acetate) and 5% solvent B (acetonitrile), followed by gradually ramping up to 100% solvent B over 24 min. The MS was operated in full-scan mode (m/z 150–1500) with a fragmentor voltage of 125 V, peak width of 0.15 min, and cycle length of 2.20 s/cycle.