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 alpha-amylase by treatment with peptide-N-glycosidase F in 18O-labeled water

Anal Biochem. 1992 Aug 15;205(1):151-8. doi: 10.1016/0003-2697(92)90592-u.


Previously, a combined use of fast atom bombardment (FAB) mass spectrometry and peptide N-glycosidase F, an enzyme that cleaves the beta-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% H2 18O, to yield a deglycosylated protein whose carbohydrate-linked Asn residues are converted to Asp partly labeled with 18O at their beta-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 beta-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 alpha-amylase, is described.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / chemistry*
  • Amino Acid Sequence
  • Asparagine / chemistry*
  • Bacillus / enzymology
  • Carbohydrate Sequence
  • Carbohydrates / chemistry*
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Glycoproteins / chemistry*
  • Glycosylation
  • Molecular Sequence Data
  • Oxygen Isotopes
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Recombinant Proteins / chemistry
  • Spectrometry, Mass, Fast Atom Bombardment
  • alpha-Amylases / chemistry*


  • Carbohydrates
  • Glycoproteins
  • Oxygen Isotopes
  • Recombinant Proteins
  • Asparagine
  • alpha-Amylases
  • Amidohydrolases
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase