Msp 96 target ground steel

Bacterial cells were spotted on a sample spot of a MALDI target plate (MSP 96 target, ground steel; Bruker Daltonics, Billerica, MA, USA) and were overlaid with HCCA matrix solution (saturated solution of α-4-cyano-hydroxycinnamic acid; Bruker Daltonics, Billerica, MA, USA) in 50% acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) and 2.5% trifluoroacetic acid solution (Sigma-Aldrich, St. Louis, MO, USA). Mass spectra profiles were acquired using a Microflex spectrometer (Bruker Daltonics, Billerica, MA, USA). The molecular ions were measured automatically in linear positive ion mode with the instrument parameters optimized for a range of 2,000–20,000 m/z. The software packages flexControl 3.0 (Bruker Daltonics, Billerica, MA, USA) and flexAnalysis 3.0 (Bruker Daltonics, Billerica, MA, USA) were used for mass spectra recording and processing. Spectra identification and analysis were carried out using the MALDI Biotyper 3.0 (Bruker Daltonics, Billerica, MA, USA). The identification was performed by comparing the obtained spectra with those in the MALDI Biotyper 3.0 library (version 3.2.1.1).

Samples for MALDI-TOF mass spectrometric analysis were prepared using HyperSep-C18-tips (Thermo Scientific, Schwerte, Germany) according to the manufacturer’s recommendations. Sample application was performed via dried-droplet on MSP 96 target ground steel (Bruker Daltonics, Bremen, Germany) with 1 µl sinapinic-matrix (45 mmol/l sinapinic acid in 70% (v/v) H₂O, 30% (v/v) acetonitrile, 0.3% (v/v) trifluoroacetic acid). After drying under vacuum, samples were analyzed using the LP-mode and 60–90% laser intensity by a Microflex device (Bruker Daltonics, Bremen, Germany). The free software mMass v. 5.5.0 was used to evaluate the mass spectra^{73 (link)}.

The colonies were directly smeared on a MALDI target plate (MSP 96 target ground steel; Bruker Dal-tonics) through a clean toothpick. Each spotted sample was overlaid with 1 μL 70% formic acid and air-dried at room temperature. Add 1 μL of HCCA matrix solution (α-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile – 2.5% trifluoroacetic acid) and air-dry for sure, then use a mass spectrometer [Bruker Microflex LT/SH “smart” MALDI-TOF MS (Bruker Daltonics, Billerica, MA, United States)] with FlexControl software (version 3.4) and MALDI Biotyper (MBT) Compass version 4.1 for analysis and identification. The mass spectrum was acquired in the mass range of 2,000 to 20,000 m/z in linear mode using MALDI-TOF MS for mass detection and analysis of bacterial proteins. The obtained spectrum was analyzed by software and then compared with all the data in the database to obtain microbial identification. The result interpretation standard was that when the identification result score of the test strain was greater than 2.00, the identification reliability was up to the strain name; scores ranging from 1.70 to 1.99 indicated the confidence level of identification up to the genus name of the strain; scores ranging from 0.00 to 1.69 indicated unreliable identification results.