To evaluate the reproducibility of the newly developed method, the entire test was repeated on a separate day. Data analysis MS spectra The MS spectra obtained from the spots overlaid with the HCCA matrix were analyzed using MALDI Biotyper 2.0 software and Bruker’s security relevant library (Bruker Daltonics). These libraries together contain 83 reference spectra (MSPs) from various Vibrio species, including three V. cholerae strains and one V. mimicus strain. For each measurement, a logarithmic score value was determined by calculating the proportion of matching peaks and peak intensities

between the test spectrum MEK inhibitor cancer and the reference spectra of the database [11, 13]. Identification at species level was based on the highest of the four logarithmic values [11]. All MS spectra obtained from spots overlaid with the FA+ matrix were analyzed using Matlab software (version R2011b). The spectra were first converted into the MZXML format using the Bruker Daltonics supplied software (CompassXport) and subsequently converted to the Matlab binary format using mzxml read procedure. Further processing was performed using the Matlab Bioinformatics toolbox (Version 4.0) routines such as resampling (msresample – mass range 10,000 to 50,000 Da and resampling

to 5,000 data points), baseline subtraction (msbackadj), alignment on a peak mass of 11974 (msalign), which was present in the MS spectra of all V. cholerae isolates, normalization (msnorm) and visualization of spectra selleckchem in a heat map. Peaks were automatically selected using standard peak selection algorithm (mspeaks – HeightFilter = 2). The highest peak in the region of 32.5 – 37.5 kDa per isolate was automatically identified. Protein identification by SDS-PAGE coupled to LC-MS/MS Viable cells of the V. cholerae isolates FFIVC129, FFIVC130, 080025/EZ, 080025/FC, 080025/FE, 080025/FI, FFIVC137 and 17/110/2006 were resuspended in 50 μl phosphate-buffered saline and mixed with 50 μl Laemmli 2x sample buffer (Bio-Rad). Samples were incubated

at 100°C for 10 minutes and analyzed by standard SDS-PAGE using a 12% polyacrylamide gel and Coomassie Brilliant Blue staining [22]. The most prominent protein bands in the mass range of 34 to 38 kDa were excised from the gel and subjected to in-gel trypsin digestion. Gel pieces were washed ZD1839 supplier with pure water, destained with three rounds of washing in a mixture of 70% 25 mM NH4HCO3/30% acetonitrile (ACN) and dehydrated by 10 minutes of incubation in 100% ACN. After removal of ACN, gel pieces were incubated in 100 mM NH4HCO3/10 mM dithiothreitol for 30 min at 56°C followed by addition of iodoacetamide to a final concentration of 55 mM and 30 min of incubation at room temperature. Gel pieces were washed in 25 mM NH4HCO3, dehydrated by incubation in 100% ACN, placed in 50 μl 100 mM NH4HCO3 containing 10 ng/ml trypsin (from bovine pancreas, Sigma-Aldrich) and incubated overnight at 37°C.