Lc ms water

Acetic, butyric and propionic acids standards and the compounds used for derivatization: 3-Nitrophenylhydrazine hydrochloride (3-NPH), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), formic acid, and pyridine were purchased from Sigma-Aldrich. LC-MS water and acetonitrile were provided by Fischer Scientific.

For database entry, strains were prepared with the formic acid extraction method to ensure a high quality of spectra. Fresh colonies were picked with a 1 μL inoculation loop and placed in 300 μL of LC–MS water (Thermo Fisher, Waltham, MA, United States). A volume of 900 μL pure ethanol (Thermo Fisher) was added to sterilize the bacteria and denature proteins. The tube was vortexed for 1 min and centrifuged at 13,000 × g for 2 min. The supernatant was discarded and the pellet was centrifuged a second time to remove ethanol residues and then dried at room temperature. Pellets were dissolved into 30 μL each of 70% formic acid (Thermo Fisher) and acetonitrile (Thermo Fisher). The solution was carefully mixed by pipetting up and down and then centrifuged at 13,000 × g for 2 min. Subsequently, 1 μL of supernatant was spotted onto an MSP 96 target polished steel BC plates in eight replicates and overlaid with 1 μL HCCA (α-Cyano-4-hydroxycinnamic acid; Bruker, Germany) matrix solution (HCCA dissolved in 50% acetonitrile, 47.5% LC–MS water and 2.5% trifluoroacetic acid) after air-drying. All V. cholerae from Zhejiang were processed in the Zhejiang Centers for Disease Control laboratory according to the above-mentioned treatment methods and carried to our laboratory for the collection of the spectra.