Acetonitrile

Fresh seeds of C. sativa var. Longma 5, which was a commercial variety from Heilongjiang Province, China (one of the major provinces in hemp cultivation), were obtained from Ms. Mu (General manager of Heilongjiang Heike Technology Co. Ltd). The chemicals including NaOH and NaCl were purchased from Hongyan Co. (Tianjin, China); NH₄HCO₃, acetonitrile, dithiothreitol, acetonitrile, formic acid, RNase-free DNase I and PBS buffer were purchased from Thermo Fisher Scientific (San Jose, CA, USA); n-hexane was purchased from Aladdin Co. (Shanghai, China); HCl was purchased from Kermel Co.(Tianjin, China); KOD enzyme was purchased from Toyobo Co. (Osaka, Japan); pGEM-T plasmid was purchased from Takara Co. (Kyoto, Japan); 5X protein loading buffer was purchased from Beyotime Co. (Shanghai, China); Coomassie brilliant blue R-250 was purchased from Solarbio Co. (Beijing, China). The kits including protein extraction kit and RNA extraction kit were purchased from Solarbio Co. (Beijing, China) and Tiangen Co. (Beijing, China), respectively. All reagents used in the study were of analytical grade or higher purity.

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.

Standards of lovastatin and simvastatin (SIMV, Internal Standard, IS) were purchased from European Pharmacopoeia (purity >98%, Strasbourg, France). Ergothioneine was purchased from Sigma Aldrich (purity >99%, St. Louis, MO, USA), while methimidazole (METH, IS) was purchased from Thermo Fisher (purity >99%, Erlenbachweg, Germany).
All standard stock solutions of lovastatin, simvastatin, and methimidazole were prepared in acetonitrile, while acetonitrile-water 7:3 (% v/v) was used to dissolve the water-soluble Ergothioneine. The prepared stock solutions were stored at −18 °C. All solvents were of an LC–MS grade. acetonitrile was purchased from Sigma Aldrich (St. Louis, MO, USA) and water was from Sharlau (Barcelona, Spain). Methanol was provided by ChemLab (Zadeglem, Belgium), while formic acid was obtained from Fisher Scientific (Hampton, VA, USA).

DB dissolution buffer (8 M urea (Sinopharm, Shanghai, China), 100 mM TEAB (Sigma, Hanover, Germany), pH 8.5) was added to each protein sample to a final volume of 100 µL. Next, 100 mM TEAB buffer (Sigma, Hanover, Germany) and trypsin (Promega, Madison, WI, USA) were added, and the samples were mixed and digested at 37 °C for 4 h. After adding additional trypsin and CaCl₂, the samples were further digested overnight. formic acid was then added to bring the pH to <3, and the samples were centrifuged at 12,000× g for 5 min at room temperature. The supernatant was gradually fed into a C18 desalination column and washed three times with washing buffer (0.1% formic acid (Thermo Fisher Scientific, Bremen, Germany), 3% acetonitrile (Thermo Fisher Scientific, Bremen, Germany)) and then eluted with elution buffer (0.1% formic acid (Thermo Fisher Scientific, Bremen, Germany), 70% acetonitrile (Thermo Fisher Scientific, Bremen, Germany)). The eluents of each sample were collected and lyophilized. Then, 100 µL of 0.1 M TEAB buffer was added to reconstitute the samples, and 41 µL of acetonitrile-dissolved TMT labeling reagent was added. The samples were mixed with shaking for 2 h at room temperature, after which the reaction was stopped by adding an equal volume of 8% ammonia. All labeled samples were then desalted and lyophilized.