Microtof 2 time of flight mass spectrometer

The EtOAcPart was also analysed by LC–ESI/TOF-MS with a 1200 Series LC-chromatograph (Agilent, Palo Alto, CA, USA) coupled with a MicrOTOF II time-of-flight mass spectrometer (Bruker Daltonics, Inc., Billerica, MA, USA). 5 µL injection was performed with an autosampler on a Poroshell EC-C18 column (100 × 2.1 mm; 2.7 µm, Agilent, Palo Alto, CA, USA). The source temperature was set at 200 °C, the drying gas (nitrogen) flow rate was 10.0 L/min and the nebulizer gas (nitrogen) pressure was 4 bar. Data were acquired in negative mode in the range of m/z 100–1500. The capillary voltage was 3.8 kV, the capillary exit voltage was −150 V, the skimmer 1 and 2 voltages were 50 V and 23 V, respectively, the hexapole 1 voltage was set to −23 V, the hexapole RF voltage was 120 Vpp, lens 1 transfer was 68 μs and lens 1 pre plus stage was 7 μs. Mass calibration was achieved by infusing ammonium formate in an isopropanol–water mixture (1:1, v/v) as an external standard. All data were analysed using Bruker Daltonics ESI Compass Data Analysis Version 4.0 SP 1 (Bruker Daltonics Inc., Billerica, MA, USA). The mobile phase consisted of spectroscopic grade methanol (B) and ultrapure water (A) containing 0.05% (v/v) formic acid. The linear gradient elution was set from 10% to 100% of B in 90 min at a flow rate of 0.3 mL/min.

Reactions were performed in a 100-μl final volume of Arr buffer (20 mM Tris-HCl [pH 7.9], 40 mM KCl, and 0.5 mM MgCl₂). M. smegmatis or M. abscessus Arr (10 μM) was mixed with Rif or KglA (100 μM) in 80 μl of Arr buffer at 37°C for 10 min. A 20-μl aliquot of NAD⁺ in Arr buffer was added (250 μM final) and incubated for 1 h at 37°C. The reaction was quenched with 500 μl of methanol (MeOH). Methanol was then evaporated under negative pressure and the reaction mixture analyzed by LC-MS. All analytical separations were performed on an Agilent 1260 HPLC instrument by injection of 1 to 5 μl of sample onto a Raptor ARC-18 column (150 mm by 2.1 mm) (Restek) or an Ultra C₄ column (150 mm by 2.1 mm) operated at 0.2 μl/min and then eluted using a 30-min linear gradient from 5% to 100% of acetonitrile. The mobile phase was supplemented with 0.1% formic acid. Mass spectra were recorded in positive-ion mode on a Bruker MicrOTOF II time-of-flight mass spectrometer.