Elsevier

Analytical Biochemistry

Volume 205, Issue 1, 15 August 1992, Pages 151-158
Analytical Biochemistry

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

https://doi.org/10.1016/0003-2697(92)90592-UGet rights and content

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% H218O, 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.

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This work was supported in part by Grants-in-Aid 02558016 and 03304028 from the Ministry of Education, Science and Culture of Japan.

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