Fourteen of the separated bands which represented major globulin subunits or polypeptides were manually excised from the gel and the gel pieces were de-stained with 50 mM NH4HCO3 in 50% (v/v) acetonitrile (ACN) and dried for 10 min with 100% ACN and then under vacuum for 30 min. Gel pieces were rehydrated in 30 lL 50 mM NH4HCO3 containing 30 ng/lL sequencing-grade trypsin (Promega) and incubated for 1 h at 4 C.