In-gel digestion was performed using a method described . Protein spots were cut from the gels, and the gel pieces were washed three times for 15 min each with 200 μl of 50 mM ammonium bicarbonate with 50% (v/v) acetonitrile and then dried under vacuum. The gel piece was rehydrated in 5 μl of sequencing-grade modified trypsin (10 ng/μl, Promega, Madison, WI) in 10 mM ammonium bicarbonate for 30 min at 4°C, and digestion was carried out for 18 h at 37°C. Peptides were extracted with 5 μl of extracting solution [50% (v/v) acetonitrile and 0.3% (v/v) trifluoroacetic acid] for 10 min by sonication.