MALDI-TOF/TOF MS Identification of Microbial Strains

MALDI–TOF/TOF MS was performed at the Synchrotron Light Research Institute, Thailand, using Autoflex^® maX (Bruker Daltonics, Bremen, Germany). Four to five pure single colonies (5–10 mg) of each microbial strain on NA medium were transferred to 1.5 mL microcentrifuge tubes and then mixed with 300 μL of HPLC–grade water. Next, 900 μL of ethanol was added, and the mixture was vortexed for 1 min. The sample solution was centrifuged at 13,500 ×g for 2 min two times. The cells were dried in a laminar air flow cabinet for 2 min, and then 5 µL of 70% formic acid was added. The sample was vortexed for 1 min. Afterwards, 5 µL of acetonitrile was added, and the solution was centrifuged at 13,000 ×g for 2 min. At this point, the clear solution was transferred to a fresh tube. Next, 1 μL of each sample was dropped on the MALDI–TOF/TOF MS target plate and left to dry; subsequently, 1 µL of HCCA matrix was dropped and left to dry. The target plates containing the samples were analyzed using MALDI–TOF/TOF MS (Autoflex^® maX, Bruker Daltonics, Karlsruhe, Germany). The spectra were recorded in the positive linear mode at the laser frequency of 60 Hz and in the mass range of m/z 2,000–10,000 Dalton. Calibration was performed using the Escherichia coli strain DH5α, which presents ribosomal protein mass with RNA and myoglobin at peaks of 5096.8, 5381.4, 6255.4, 7274.5, 10,300.1, 13,683.2, and 16,952.3 m/z. Two–thousand shots of the mass spectrum profile data were collected from each sample. The mass spectrum profiles were processed with Flexcontrol v.3.4 (Karlsruhe, Germany). The protein mass fingerprints were analyzed at a molecular weight tolerance of 300 ppm. The resulting data were imported to MALDI BioTyper v.4.0 (Karlsruhe, Germany) to compare the sample mass fingerprints to the database (Elshafie et al., 2015 (link)).