New insights in Rapana venosa hemocyanin N-glycosylation resulting from on-line mass spectrometric analyses

• Published in: Glycobiology (Oxford) Vol. 17; no. 2; pp. 141 - 156
• Main Authors: Sandra, Koen, Dolashka-Angelova, Pavlina, Devreese, Bart, Van Beeumen, Jozef
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-02-2007; Oxford Publishing Limited (England)
• Subjects: Amino Acid Sequence; Animals; capillary electrophoresis-mass spectrometry; Electrophoresis, Capillary; Gastropoda - metabolism; Glycoside Hydrolases - chemistry; Glycosylation; hemocyanin; Hemocyanins - chemistry; Hemocyanins - metabolism; MALDI-TOF; Molecular Sequence Data; Online Systems; Polysaccharides - analysis; Pyrazoles - chemistry; Pyrenes - chemistry; Rapana venosa; Sequence Analysis, Protein; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; TOF; Trypsin - chemistry; glycosylation; TOF; MALDI-TOF; capillary electrophoresis-mass spectrometry; hemocyanin
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwl063

The N-glycosylation of structural unit 1 of Rapana venosa hemocyanin was studied. Enzymatically liberated N-glycans were analyzed by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis (CE)-MS following 8-aminopyrene-1,3,6-trisulfonate labeling and labeling with 3-aminopyrazole, a new dedicated sugar reagent. Structural information was obtained by exoglycosidase sequencing, on-line MS/MS, permethylation, and amidation. A mixture of high-mannose and complex glycans with so far unknown and unusual acidic terminal structures was revealed. As the hemocyanin protein sequence is currently unknown, de novo sequencing of the glycopeptides had to be carried out. The N-glycans were therefore enzymatically removed with simultaneous partial (50%) 18O-labeling of glycosylated asparagine residues prior to proteolysis. Following nano-liquid chromatography-MALDI-TOF-MS, the originally glycosylated peptides could be revealed and their sequences determined by MS/MS. The site occupancies were subsequently elucidated by precursor ion scanning of the intact glycopeptides using a Q-Trap mass spectrometer.