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Glycobiology Advance Access originally published online on October 26, 2006
Glycobiology 2007 17(2):141-156; doi:10.1093/glycob/cwl063
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Koen Sandra2,3, Pavlina Dolashka-Angelova4, Bart Devreese3 and Jozef Van Beeumen1,3

3 Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium
4 Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

1 To whom correspondence should be addressed; Tel: +32 9 264 5109; Fax: +32 9 264 5338; e-mail: jozef.vanbeeumen{at}ugent.be

Received on April 27, 2006; revised on October 16, 2006; accepted on October 20, 2006

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.

Key words: capillary electrophoresis-mass spectrometry / glycosylation / hemocyanin / MALDI-TOF / TOF / Rapana venosa

2 Present address: Peakadilly nv., Technologiepark 4, VIB Bio-incubator, B-9052 Zwijnaarde/Ghent, Belgium, e-mail: koen.sandra{at}peakadilly.com

None declared.


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