Sonication vials

The ATCC/DSMZ type strains and clinical isolates of M. abs complex were cultured on 7H11 agar plates for 4-5 days in a CO₂ incubator maintained at 37°C. Approximately 1 µL loopful of colonies was transferred into sonication vials (Thermo Fisher Scientific, proprietary). The cells were then dispensed in a pre-incubation solution containing alcohol (Thermo Fisher Scientific, proprietary) at room temperature (RT, 20-25°C). After a short centrifugation step (12,000 x g for 2 minutes at RT) the supernatant was discarded and to the pellet 100 µL of incubation solution containing formic acid and acetonitrile (Thermo Fisher Scientific, proprietary) was added. The cell lysate was incubated for 20 minutes (vortexed once at 10 minutes for 2 seconds). Sonication was then performed for one minute at 50% amplitude, then 100 µL dilution buffer containing acetonitrile (Thermo Fisher Scientific, proprietary) was added to the lysed cells and centrifuged at 12,000 × g at RT for 5 minutes. The supernatant was collected in low protein binding Eppendorf (LBE) tubes and can be stored at -80°C until its LC-MS analysis is performed.

ATCC strains and clinical isolates of M. tuberculosis complex were cultured on 7H11 agar plates for approximately 18-24 days in a CO2 incubator maintained at 37°C in a BSL3 laboratory. A 1 µL loopful of colonies was transferred into sonication vials (Thermo Fisher Scientific, proprietary). Subsequently the pre-incubation solution containing alcohol (Thermo Fisher Scientific, proprietary) was added to the cells at room temperature (RT, 20-25°C) and following a short centrifugation step (12,000 x g for 2 minutes at RT) the supernatant was discarded and then the pellet was suspended into 100 µL of incubation solution containing formic acid and acetonitrile (Thermo Fisher Scientific, proprietary). The cell lysate were incubated for 20 minutes (vortexed once at the 10-minute mark for 2 seconds), followed by sonication for one minute at 50% amplitude. Cells were diluted with 100 µL dilution buffer containing acetonitrile (Thermo Fisher Scientific, proprietary) and centrifuged for 5 minutes at 12,000 × g at RT. The supernatant was collected in low protein binding (LBE) Eppendorf tubes and stored at -80°C if not immediately used for LC-MS analysis.