Maldi tof tof ms 4800 proteomics analyzer

Selected protein spots of interest were excised and pooled from the stained 2-DE gels and digested with solution of sequencing-grade modified trypsin (Promega, Madison, USA). The peptides released from the gel plugs were then sent to the Australian Proteome Analysis Facility, Australia for further analysis using MALDI-TOF/TOF MS (4800 Proteomics Analyzer, AB Sciex). The subsequent search settings were used: carboxymidomethylation of cysteine was fixed modification and oxidation of methionine was selected as variable modification; maximum number of missed cleavages: 1; peptide tolerance (peptide tol): ±0.6; peptide charge: +1.

Samples were separated by SDS-PAGE (12.5%) and the gel was stained with Coomassie brilliant blue (CBB). Gag was excised from the stained gel and digested with trypsin in 50 mM NH₄HCO₃ (pH 8.0) for 12 h at 37°C. Phosphopeptides were enriched using Titansphere® Phos-TiO Kit (GLsciences, Tokyo, Japan), in accordance with the manufacturer’s instructions. The enriched phosphopeptides were then analyzed by MALDI-TOF/TOF-MS (4800 proteomics analyzer, AB SCIEX, Foster City, CA). The resulting raw MS spectrum was processed using the 4000 Series Explorer Software (AB SCIEX, Framingham, MA) to generate Mascot generic format. The obtained MS and MS/MS data were then searched against the SwissProt database (January, 2013; 538849 sequences) using Mascot version 2.4.1 software (Matrix Science, London, UK), to identify proteins and protein modification. The search parameters were as follows: trypsin digestion with two missed cleavages permitted, variable modifications (oxidation of Met and phosphorylation of Ser, Thr, and Tyr), peptide mass tolerance for MS data ±0.15 Da, and fragment mass tolerance ±0.3 Da. Phosphopeptides were determined primarily using the Mascot program and were confirmed manually through raw MS/MS sequence data checking for the neutral loss of the phosphate group (−98).