Evaluation of proteolytically released carbohydrate-containing peptides of bovine prothrombin fragment 1 using electrospray ionization mass spectrometry and capillary electrophoresis

Described here is a method used to determine the sialic acid content of the structurally distinct carbohydrates attached at N77 and N101 of bovine prothrombin fragment 1. The protein is proteolytically cleaved with alpha-chymotrypsin to release two glycopeptides, each containing one of the two glycosylation sites. Electrospray ionization mass spectrometry and capillary electrophoresis are used to determine the structural difference between the carbohydrates of each of these glycopeptides. N77 bears a carbohydrate containing as many as 4 sialic acid (N-acetylneuraminic acid) residues, whereas the carbohydrate attached at N101 may carry as many as 5 sialic acid residues.