Each spectrum was created with the software Flex Control

(Version 3.3) either in an automatic mode with variable laser power or manually with a laser power set between 29–33%. For each spectrum a total of 240 shots were summed up. Before each measurement, the instrument was calibrated using the bacterial test standard (BTS), an Escherichia coli DH5alpha extract, spiked with two additional proteins (RNase A and myoglobine) provided by Bruker Daltonik GmbH (Bremen, Germany). Preparation of the BTS and calibration were performed following the manufacturer’s instructions. Calibration was buy QNZ successful when proteins of the mass spectra were in a range of ± 300 ppm (parts per million). Protein reference spectra creation and MALDI-TOF MS measurements For the 28 leptospiral reference strains (Table 1) reference spectra, in the following called MSPs (main spectral projections), were created independently in two different laboratories. Main spectra represent

individual protein spectra for one bacterial strain. To achieve representative results, at least 20 individual spectra were used to create a single MSP as proposed by Bruker Daltonik GmbH (Bremen, Germany). Each sample was spotted on eight positions of the target and 24 to 30 raw spectra of the leptospiral strain and one spectrum of the bacterial test standard were measured automatically with the Flex control software. Spectra were analyzed with the Flex Analysis software (Version 3.3). The BTS was used for internal calibration. In a second step the PF-3084014 ic50 uniformity of the created spectra sets HDAC activation was visually checked in a mass range of 3,000 Da to 10,000 Da. Spectra with peaks outside the allowed average were removed. Modified spectra were loaded into the MALDI BioTyper™ 3.0 Version (Bruker Daltonik GmbH, Bremen, Germany). Software settings for MSP creation were set to: maximal mass error of each single spectrum: 2,000; desired mass error for

the MSP: 200; desired peak frequency minimum (%): 25; maximal desired peak number of the MSP: 70. Reference spectra were created automatically by the software and all created spectra were added to the main spectra library as unassigned MSPs. The created reference spectra were evaluated based on measurements with the defined strains (see Table 1) and, additionally, with 16 field isolates (Table 2). Each strain was prepared using the Ribonuclease T1 ethanol/formic acid extraction and spotted on the target. Each spot was measured twice in both automatic and manual modes on different target spots. Table 2 16 Leptospira field isolates identified by MALDI-TOF MS measurements and 16S rRNA sequencing genomospecies serogroup serovar strain number origin L. borgpetersenii Sejroe Saxkoebing LGL 489 corpus vitreum, horse L. interrogans Australis Australis LGL 537 corpus vitreum, horse L. interrogans Australis Bratislava LGL 538 corpus vitreum, horse L. interrogans Icterohaemorrhagiae Icterohaemorrhagiae LGL 113 human urine L. interrogans Icterohaemorrhagiae Icterohaemorrhagiae LGL 535 human urine L.