Denovo explorer software

The peptide sequences were predicted by Matrix Assisted Laser Desorption/Ionization Time Of Flight Tandem Mass Spectrometry (MALDI-TOF/MS) method using a 4700 Proteomics Analyzer with Denovo Explorer software (Applied Biosystems, Carlsbad, CA, USA). The predicted peptide sequences from WSP were calculated by PeptideCutter (https://web.expasy.org/peptide_cutter/).

For the identification of the target protein, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used. Proteins were visualized with CBB-G250 after SDS-PAGE, and then the target bands were excised from the gel and washed with 25 mM NH HCO , 40 % (v/v) ethanol five times. The gel was cut into pieces and dehydrated with 1 ml of acetonitrile and dried in vacuo. Following this, 0.1 µg/ml trypsin in 25 mM NH HCO was added to the gel pieces and incubated at 37 °C for 16 h. Peptide fragments were extracted from the gel pieces with 50 % (v/v) acetonitrile, 5 % (v/v) trifluoroacetic acid for 30 min. The extracts were dried in vacuo, dissolved in 5 µl of 50 % (v/v) acetonitrile, 0.1 % (v/v) trifluoroacetic acid, and subjected to an ABI 4700 proteomics analyzer (Applied Biosystems). Mass fingerprints of tryptic peptides dissolved in 5 µl of 50 % (v/v) acetonitrile, 0.1 % (v/v) trifluoroacetic acid were generated by MALDI-TOF-MS using an Applied Biosystems 4700 Proteomics Analyser with TOF/TOF optics in the MS mode. A Nd:YAG laser (355 nm) was used to irradiate the sample. The spectra were acquired in reflection mode in the mass range 700-3200 Da. Amino-acid sequences of the fragments were determined in MS/MS mode with DeNovo Explorer software (Applied Biosystems).