0, Bruker Daltonik GmbH, Bremen, Germany) following

the guidelines of the manufacturer. Each sample was spotted onto six target spots of a steel MALDI target plate. Spectra were acquired with an UltraflexTM I instrument (Bruker Daltonik GmbH) in the linear positive mode in the range of 2,000 to 20,000 Da. Acceleration Voltage was 25 kV and the instrument was calibrated in the range between 3,637.8 and 16,952.3 Da with Bacterial Test Standard calibrant (BTS, Bruker Daltonik GmbH). Four single mass spectra with 500 shots each were acquired IACS-010759 from each spot and a selleck reference spectrum calculated from the 24 single spectra. Reference spectra contained the parameters peak mass and intensity and additional information on the reproducibility of the mass peaks, i.e. the frequency of occurrence of every peak in the underlying 24 single spectra. Reference spectra were generated within the mass range of 2,000

to 20,000 Da with the default parameter settings in the MALDI Biotyper software. The Captisol molecular weight number of peaks was limited to 100 per reference spectrum and all peaks of a reference spectrum were normalized to the most intense peak with an intensity of 1.0. The minimum frequency of occurrence within the 24 single spectra was set to 50% for every mass. Peaklists of reference spectra were exported for further evaluation in the statistical programming language R. To test the inter-laboratory variation and the robustness of the classification by using MALDI Biotyper software, a set of B. mallei and B. pseudomallei Interleukin-3 receptor test samples from a second laboratory (Table 3) was queried against the reference spectra set described above. These spectra were recorded at the Bundeswehr Institute of Microbiology with an Autoflex mass spectrometer (Bruker Daltonik GmbH, Bremens). Spectra were generated for the test set in the same way as

for the reference set. A query of all test samples was performed and the resulting scores were transferred into a data matrix, the ‘score matrix’, in which every column represented a member of the reference set and every line a test sample. The power of certain combinations of representatives of the two classes within the reference set to discriminate samples of the test set was tested as follows: the columns representing the combination of reference spectra to be evaluated were selected from the score matrix and every member of the test set was classified by assigning it to the class of the member of the reduced reference set with the highest score. The number of correct and incorrect assignments was then calculated for the test set. This procedure simulates a MALDI Biotyper query with a reduced number of spectra in the reference database.