Protein identification through tandem mass spectrometry

The selected spots were cut from 2-DE gels and decolored by bleaching solution (50 % 25 mM NH₄HCO₃ and 50 % acetonitrile) in EP tubes. After the protein spots colorless, the decoloring liquid was discard and 100 μl acetonitrile was add to the EP tubes. After samples turned white, dry treatment was performed for at least 30 min. The dry samples were digested with 7 μl diluted solvent (trypsin enzyme solution diluted with 25 mM NH₄HCO₃, the final concentration 15 ng/μl), and incubated at 37 °C for at least 16 h. Subsequently, the peptides were extracted with 5 % trifluoroacetic acid (TFA), 50 % acetonitrile and 45 % water at 37 °C for 1 h. Extracts were dried using a vacuum dryer. The dried peptide mixtures were completely dissolved in 2 μl solution containing 0.1 % TFA mixed with 1 μl TFA, 500 μl acetonitrile solution and 499 μl double distilled water.

Tryptic peptides were analyzed with a MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA). All the MS/MS spectra were searched in the NCBI non-redundant green plant database. The peptide mass tolerance was 100 ppm, the fragment mass tolerance were 0.2 Da, allowed one missed cleavage. Carbamidomethyl (Cys) and oxidation (Met) were specified as variable modifications. Only MASCOT scores more than 65 (p < 0. 05) were accepted.