Abstract

European Journal of Mass Spectrometry
Volume 14 Issue 6, Pages 345–354 (2008)
doi: 10.1255/ejms.979

Deciphering Dorin M glycosylation by mass spectrometry

Petr Man,^a* Vojtěch Kovář,^b Ján Štěrba,^b Martin Strohalm,^a,c Daniel Kavan,^a Petr Kopáček,^b Libor Grubhoffer^b and Vladimír Havlíček^a
aInstitute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4, Czech Republic. E-mail: pman@biomed.cas.cz
^bInstitute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, v.v.i., and Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic
^cDepartment of Biochemistry and Microbiology, Institute of Chemical Technology, 166 28 Praha 6, Czech Republic

The soft tick, Ornithodoros moubata, is a vector of several bacterial and viral pathogens including Borrelia duttoni, a causative agent of relapsing fever and African swine fever virus. Previously, a sialic acid-specific lectin Dorin M was isolated from its hemolymph. Here, we report on the complete characterization of the primary sequence of Dorin M. Using liquid chromatography coupled to mass spectrometry, we identified three different glycopeptides in the tryptic digest of Dorin M. The peptide, as well as the glycan part of all glycopeptides, were further fully sequenced by means of tandem mass spectrometry (MS²) and multiple-stage mass spectrometry (MS³). Two classical N-glycosylation sites were modified by high-mannose-type glycans containing up to nine mannose residues. The third site bore a glycan with four to five mannose residues and a deoxyhexose (fucose) attached to the proximal N-acetylglycosamine. The microheterogeneity at each site was estimated based on chromatographic behavior of different glycoforms. The fourth, a non-classical N-glycosylation site (Asn–Asn–Cys), was not glycosylated, probably due to the involvement of the cysteine residue in a disulfide bridge.

Keywords: glycosylation, tandem mass spectrometry, liquid chromatography, lectin, soft tick

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