Microflex lt maldi tof ms instrument

Mass spectra were collected using a Microflex LT MALDI-TOF MS instrument (Bruker Daltonics, Bremen, Germany) operated by FlexControl v3.4. A bacterial colony was directly smeared on a steel MALDI plate without protein extraction. Dried samples were overlaid with 1 μl of formic acid and 1 μl of CHCA matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile with 2.5% trifluoroacetic acid), sequentially. The Bruker Bacterial Test Standard was used for mass calibration. The acquired raw spectra were exported from FlexAnalysis (Bruker Daltonics). Spectra were obtained in the range of 1960 to 20000 m/z.

Protein (0.5 μL) obtained by centrifugation was uniformly coated on the 96-well target plate and dried at room temperature. Each well was then overlaid with 0.5 μL α-cyano-4-hydroxycinnamic acid (Fluka) saturated with a 50:47.5:2.5 mixture of acetonitrile:water:trifluoroacetic acid (Sigma, Ameican). After the sample was dried, the target plate is placed into the Microflex LT MALDI-TOF MS instrument (Bruker Daltonics, Germany) for detection. The parameters of the instrument were set in the positive linear operation mode, the mass range collected by MALDI-TOF MS was 2,000~20,000, the acceleration voltage was 20 kV, the extraction voltage was 18.6 kV, the focusing voltage was 6.5 kV, and the extraction delay time was 150 ns. Calibration was performed with Protein standard I. Mass spectra were analyzed by Flex Control 3.0.

Identified CoNS strains were selected for species confirmation using MALDI-TOF MS [16 ]. Each strain was sub-cultured on sheep blood agar and incubated overnight at 37 °C to obtain a single pure colony for MALDI-TOF MS analysis. Briefly, a single pure colony of each strain was spotted on a ground steel MALDI target plate in duplicate and allowed to dry. To each spot, 1 μL of the matrix (Bruker Daltonik GmbH, Bremen, Germany), containing a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile (Sigma-Aldrich) and 2.5% trifluoro-acetic acid (Sigma-Aldrich) was added to the spot and was allowed to dry. Thereafter, the spotted plate was placed for analysis in Microflex LT MALDI-TOF MS instrument (Bruker Daltonik GmbH) with the following parameter settings; Ion Source (IS) 1 20 kV, IS2 18.5 kV, lens 8.5 kV, pulsed ion extraction 250 nucleotides with no gating. Spectra calibration and the measurements were carried out for species identification with reference strains, and the highest score for each species was selected according to the manufacturer’s instructions.