Flexcontrol software v3

For MALDI-TOF MS, in-gel digestion of sample was performed. Tryptic peptides were analyzed in an ultrafl Xtreme^™ MALDI TOF/TOF device (Bruker, Bremen, Germany) using Flex Control software v3.3 (Bruker). Protein identification was performed using Mascot search engine (Matrix Science, London, UK), using the following parameters: monoisotopic mass accuracy, fragment mass tolerance ± 0.5 Da; missed cleavages 0, allowed variable modifications, oxidation (Met), and carbamidomethylation (Cys). Positive identification was retained with MASCOT scores above the threshold level (P<0.05).

All MALDI-TOF experiments were performed at CAARU, Sultan Qaboos University, on UltraFlextreme (Bruker Daltonics, Bremen, Germany) operating in positive reflectron mode in the m/z range of 50–2000 Da. Stainless steel MTP 384 target plate was used for all the molecular weight analysis. Dihydroxy benzoic acid (DHB) dried droplet protocol described in Bruker manual was employed for sample preparation and spotting. Two micro liter of 2, 5-DHB matrix (20 mg/ml) in TA 30 (30:70 v/v ACN:TFA 0.1%TFA) was premixed with 2 μl of the sample solution. One micro liter of the mixture was applied to the ground steel target plate, dried at room temperature. The spectra were acquired using FlexControl software v3.3 (Bruker Daltonics, Bremen, Germany). A SmartBeam-II laser, set at a frequency of 1000 Hz, was used for ionization. The laser strength was optimized at 25–35%. A summed spectrum was obtained for each MALDI-spot. Peaks were detected using the SNAP peak detection algorithm and a baseline subtraction was carried out using “TopHat” algorithm. The MALDI-TOF spectra were externally calibrated using a commercially available peptide mix (peptide calibration standard II, Part-No #222570, Bruker Daltonics, Germany). FlexAnalysis Software v3.3 (Bruker Daltonics) was used for visualization and initial data processing.