A 0.8 μl aliquot of each peptide mixture was deposited onto a 386-well OptiTOF™ Plate (Applied Biosystems, Framingham, MA, USA) and allowed to dry at room temperature. A 0.8 μl aliquot of matrix solution (3 mg/mL CHCA in MALDI solution) was then added onto dried digest and allowed to dry at room temperature. MALDI peptide mass fingerprinting, MS/MS analysis
and database searching For MALDI-TOF/TOF analysis, samples were automatically acquired in an ABi 4800 MALDI TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA, USA) in positive ion reflector mode (ion acceleration voltage was 25 kV for MS acquisition and 1 kV for MSMS) and the spectra were stored into click here the ABi 4000 Series Explorer Spot Set Manager. PMF and MSMS fragment ion spectra were smoothed and corrected to zero baseline using routines embedded in ABi 4000 Series Explorer Software v3.6. Each PMF spectrum was internally calibrated with the mass signals of trypsin autolysis ions to reach a typical mass measurement accuracy of <25 ppm. Known Batimastat in vitro trypsin and keratin mass signals, as well as potential sodium and potassium adducts (+21 Da and +39 Da) were removed from the peak list. To submit the combined PMF and MS/MS data to MASCOT software v.2.1 (Matrix Science, London, UK), GPS Explorer v4.9 was used, searching in the non-redundant
NCBI protein database. LC-ESI MS/MS analysis In some specific cases, alternative proteomic techniques were employed to confirm and improve protein identifications. For this purpose, we made use of liquid chromatography coupled to electrospray ion-trap mass spectrometry tandem MS (LC ESI-MS/MS). This was done using an Ultimate 3000 nano LC (Dionex, Amsterdam, Astemizole The Netherland) and a 75 micrometer I.D, 100 mm reversed-phase column, at a 300 nL/min flow, coupled to a Bruker HCT Ultra ion-trap mass spectrometer (Bruker Daltonics, Bremen,
Germany) working in dynamic exclusion mode. Database Search For protein identification, LC ESI MS/MS spectra were transferred to BioTools 2.0 interface (Bruker Daltonics) to search in the NCBInr database using a licensed version of Mascot v.2.2.04 search engine (http://www.matrixscience.com; Matrix Science, London, UK). Search parameters were set as follows: carbamidomethyl cystein as fixed modification by the treatment with iodoacetamide, oxidized methionines as variable modification, peptide mass tolerance of 0.5 Da for the parental mass and fragment masses and 1 missed cleavage site. In all protein identifications, the probability Mowse scores were greater than the minimum score fixed as significant with a p-value minor than 0.05. Selected proteins were based on that who exhibited higher Mascot score and sequence coverage. A total of thirty-three different proteins showing differential expression pattern between polyP+ and polyP- this website strains (three independent replicates) were selected.