Figure 2. RP-HPLC of neutral N-glycans of honeybee venom.

N-glycans released by the combined use of PNGase F and Ar were subject to solid phase extraction, whereby the neutral-enriched fraction was eluted with 40% acetonitrile, prior to fluorescent labelling and chromatography on an RP-amide column; each fraction was collected and subject to MALDI-TOF MS. The annotations in the Symbolic Nomenclature for Glycans (see also key in grey box) are based on elution time, MS/MS and digestion data (see examples in Supplementary Figures 2 and 3). The column was calibrated in terms of glucose units. Phosphoethanolamine (PE)- or α-GalNAc-modified glycans in this pool are highlighted respectively in blue or green boxes, structures previously found on honeybee venom phospholipase A₂ and hyaluronidase are in light grey boxes and those hybrid or biantennary forms reported by us in royal jelly in light yellow boxes; seven different isomers of Hex₃HexNAc₄Fuc₂ are indicated by the m/z 1687 values in red. Due to their low abundance, neither the biantennary and Man_4-5GlcNAc₂-based hybrid glycans nor the PE-, β1,3-Gal and α1·4· alNAc-modified antennae were previously detected in honeybee venom.