MASS-SPECTROMETRIC CHARACTERIZATION AND GLYCOSYLATION PROFILE OF BOVINE PANCREATIC BILE SALT-ACTIVATED LIPASE

We developed a procedure for the large scale isolation of bovine bile salt-activated lipase (BAL) for its crystallization [Wang, X. et al. ( 1997) Structure 5, 1209-1218] and also carried out a study on the mole cule's glycosylation profile for a better deduction of the structure o f the enzyme, Mass spectrometric analysis of the CNBr-generated peptid es indicated that only one (Asn-361) of the two potential N-glycosylat ion sites (Asn-187 and Asn-361) with NXT motif is glycosylated. The an alysis of the isolated CNBr peptide containing Asn-361 showed that it existed in three glycoforms in a ratio of 1.0:2.8:1.0, with oligosacch aride moieties weighing 1900.1, 2045.2, and 2336.4 Da, respectively. T he major oligosaccharide chain contained annose:galactose:N-acetylgluc osamine:fucose:sialic acid in a molar ratio of 2:2:4:2:1. It was also determined that the potential O-glycosylated peptide (CB13) is not O-g lycosylated and, in addition, it was found that there was microheterog eneity in the C-terminus of the isolated bovine BAL. The results obtai ned from this mass spectrometric study combined with the X-ray crystal lographic studies provide more precise structural information on BAL. The procedure described here for the mass spectrometric analysis of CN Br-generated peptides also has general applicability for analysis of t he glycosylation profile of glycoproteins and the C-terminal peptide s tructure of proteins. (C) 1998 Academic Press.