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New insights in Rapana venosa hemocyanin N-glycosylation resulting from on-line mass spectrometric analyses
Sandra, K.; Dolashka-Angelova, P.; Devreese, B.; Van Beeumen, J. (2007). New insights in Rapana venosa hemocyanin N-glycosylation resulting from on-line mass spectrometric analyses. Glycobiology 17(2): 141-156. dx.doi.org/10.1093/glycob/cwl063
In: Glycobiology. OXFORD UNIV PRESS INC: Cary. ISSN 0959-6658; e-ISSN 1460-2423, more
Peer reviewed article  

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Keywords
    Rapana venosa (Valenciennes, 1846) [WoRMS]
    Marine
Author keywords
    capillary electrophoresis-mass spectrometry; glycosylation; hemocyanin;MALDI-TOF; TOF; Rapana venosa

Authors  Top 
  • Sandra, K.
  • Dolashka-Angelova, P.
  • Devreese, B.
  • Van Beeumen, J.

Abstract
    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.

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