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.
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.
Gonzalez
J
J
Institute for Protein Research, Osaka University, Japan.
Takao
T
T
Hori
H
H
Besada
V
V
Rodriguez
R
R
Padron
G
G
Shimonishi
Y
Y
D000581:Amidohydrolases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000737:chemistry; D016339:Spectrometry, Mass, Fast Atom Bombardment; D000516:alpha-Amylases / Q000737:chemistry*
D002241:Carbohydrates; D006023:Glycoproteins; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D001216:Asparagine; D000516:alpha-Amylases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase