Protein bands of interest were excised from the gel. The excised gel pieces were washed first with dd-H20 and subsequently with washing solution I (50% Methanol, 25mM Ammonium bicarbonate, pH 8.3), and washing solution II (50% Acetonitrile, 25mM Ammonium bicarbonate, pH 8.3). The washed gel pieces were finally dehydrated with 100% acetonitrile and dried under speed-vac. The dried gel pieces were either underwent trypsin digestion or kept at -80°C until they were treated with trypsin for the MS -peptide analysis. In brief, the gel pieces were incubated with appropriate amount of trypsin (Modified Trypsin Gold, Promega, Madison, WI) in proteaseMAX surfectant (Promega, Madison, WI) at 37°C for 2~ 3 hours. After incubation, the digested peptides were extracted with 2.5% of trifluoroacetic acid. The extracted peptides were further purified and concentrated by ZipTip, a C18 micro-reverse phase resin tip (Millipore, Billerica, MA) according to the manufacturer’s protocol.
Extracted peptides were then analyzed by 4800 MALDI Tof/Tof tandem mass spectrometer (AB Sciex, Framingham, MA) with MS/MS tandem mode. Protein identifications were performed using Mascot search engine (Matrix Science Inc, Boston, MA) against Swiss Pro or NCBI protein database.
Extracted peptides were then analyzed by 4800 MALDI Tof/Tof tandem mass spectrometer (AB Sciex, Framingham, MA) with MS/MS tandem mode. Protein identifications were performed using Mascot search engine (Matrix Science Inc, Boston, MA) against Swiss Pro or NCBI protein database.