Briefly, 1 mL of saliva sample with or without PI was concentrated with a 10 kDa membrane cut-off filter (Millipore). The fractions with high-molecular-weight proteins were separated check details on 1D SDS-PAGE (Novex Bis–Tris 4-12% gel; Invitrogen)

and stained with Coomassie Blue. The protein gel bands were excised from the 1D SDS-PAGE and subjected to in-gel reduction, alkylation, and trypsin digestion. The digestion was performed for 16 h at 37 °C. The peptides generated were extracted with 50% acetonitrile, washed twice with a solution containing 0.1% TFA and dried with a Speed-Vac. The dried peptide mixture was subjected to LC-MS/MS analysis. For LC-MS/MS analysis, the peptide mixture was separated by a 60-min gradient elution with the Dionex U3000 capillary/nano-HPLC system (Dionex, Sunnyvale, CA) at a flow rate of 0.25 μL min−1 directly interfaced with a Thermo-Fisher LTQ-Orbitrap mass spectrometer (Thermo-Fisher, San Jose, CA) operated in the

data-dependent scan mode. The analytical column was a homemade fused silica capillary column (75 μm i.d., 100 mm length; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 A, 5 μm; Varian, Palo Alto, CA). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile Ensartinib nmr and 0.1% formic acid. The 60-min gradients at 0.250 μL min−1 flow rate for solvent B increased from 0% to 55% in 30 min and then to 80% in 10 min. The experiment consisted of a single full-scan mass spectrum in the Orbitrap (400–1600 m/z, 30 000 resolutions), which was followed by six data-dependent MS/MS scans in the ion trap at 35% normalized collision energy. Data were analyzed using mascot software and manual inspection. The fraction with low-molecular-weight species was directly

new analyzed by LC-MS/MS using the method described above with an LTQ-Orbitrap mass spectrometer (Thermo-Fisher). Data management and analyses were performed using spss 17.0 software (SPSS Inc., Chicago, IL). All cultivable bacterial data were compiled and logarithmically transformed to normalize the variance distribution. Correlation analyses were performed to determine the correlation coefficients of the mean bacterial levels in the samples with and without PI addition. For DGGE profile analysis, levels of similarity between fingerprints were calculated according to the Dice coefficient. Dendrograms were constructed from the average matrix using the unweighted pair group method by means of arithmetic averages. Differences in mean bacterial counts (log10 value), the number of detected DGGE bands, and the degree of similarity were evaluated using the paired t-test. All P values <0.05 were two-tailed and considered significant. Based on conventional culturing techniques, the log10 values of the total cultivable bacteria in saliva with PI were similar to those of saliva without PI.