Two-dimensional gel excision, Tryptic Digestion, and Desalting

All the the immunodominant spots on the PVDF membranes were excised from 2D gels from the preparative gels. Subsequently, the protein spots were destained for 20 min in 30 mM potassium ferricyanide/100mM sodium thiosulfate (1:1 v/v) and washed with Milli-Q waters. The spots were incubated in 0.2 M NH₄HCO₃ for 20 min and then lyophilized. Each spot was digested overnight in 12.5 ng/μl trypsin in 25 mM NH₄HCO₃. The peptides were extracted three times with 60% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA). The extracts were pooled and dried completely by a vacuum centrifuge.