Montage in gel digestzp kit

Protein spot digestion and protein identification were performed as previously described38 (link). The target protein spots in the 2-DE gels were selected and submitted to trypsin digestion using the Montage In-Gel DigestZP Kit (Millipore, Bedford, MA, USA). A Bruker REFLEX III MALDI-TOF-MS instrument (Bruker Daltonics, Bremen, Germany) was used for the analysis. The mass accuracy of the mass spectral peak was calibrated using the auto-digestion peak of trypsin. The mass spectrometry data were subjected to NCBI (version 18/09/2010) database searches using Mascot software (version 2.2.04; Matrix Science, London, UK). According to the input protein isoelectric point, molecular weight range, and PMF data, searches for proteins with matching parameters were performed in the database. The following procedure for database searching is provided at http://www.matrixscience.com. MASCOT search parameters were as follows: no molecular weight restriction, 2 miscleavages allowed, enzyme = trypsin, variable modifications = oxidation (M)/carbamidomethyl (C), peptide tolerance = 0.3 Da, peptide charge = 1+. In the whole experiment, masks and gloves were worn to reduce keratin contamination.

For the mass spectometry analysis, several spots were selected based on diffrrences in intensity between spots grown under pH 4.0, pH 9.0, or M9 medium, and spots from the reference gel (grown under pH 7.0.). The selected spots were excised from the polyacrylamide gels, and disrupted in order to digest proteins prior to mass spectrometry. In brief, the extracts were digested with Trypsin Porcine Pancreas (Sigma) using the Montage In-Gel DigestZP Kit (ZipPlate, Millipore, USA) following the manufacturer’s recommendations.