A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: Identification of the positions of carbohydrate-linked asparagine in recombinant α-amylase by treatment with peptide- N-glycosidase F in 18O-labeled water

Abstract

Previously, a combined use of fast atom bombardment (FAB) mass spectrometry and peptide N-glycosidase F, an enzyme that cleaves the β-aspartylglycosylamine linkage of Asn-linked carbohydrates, was successfully applied to identification of N-glycosylation sites in a glycoprotein with the known or DNA-derived sequence ( S. A. Carr and G. D. Roberts, 1986, Anal. Biochem. 157, 396–406). Here, we extended the method for easier identification of N-glycosylation sites in a glycoprotein even with unknown sequence. The glycoprotein is digested with peptide- N-glycosidase F in buffer containing 40 at% H 2 18O, to yield a deglycosylated protein whose carbohydrate-linked Asn residues are converted to Asp partly labeled with 18O at their β-carboxyl group during this digestion. The deglycosylated protein is further digested with proteolytic enzymes in an appropriate buffer prepared with normal water, and then peptides are separated on a reversed-phase column by HPLC. Peptides in which carbohydrate-linked Asn has been converted to Asp show a pair of signals ([ M + 1] + and [ M + 3] +) in FAB mass spectra due to the partial incorporation of 18O into the β-carboxyl groups of Asp residues, while the other peptides show normal isotopic ion distributions. Thus, both formally N-glycosylated peptides and, using collision-induced dissociation analysis, N-glycosylation sites can be identified. The application of the present method to the determination of N-glycosylation sites in a recombinant glycoprotein, Bacillus licheniformis α-amylase, is described.