Figure 1. RP-HPLC of neutral N-glycans of male honeybee larvae.

PNGase F-released N-glycans 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 (m/z values for [M+H]⁺ being indicated); the glycans in each fraction are shown in order of occurrence with the most dominant glycan uppermost. The annotations in the Symbolic Nomenclature for Glycans (see also key in grey box) are based on elution time, MS/MS and digestion data in comparison to recently-published data on royal jelly, mosquito and moth N-glycans; for some simple glycans, a table of elution times in comparison to previous studies is given in Supplementary Table 2, whereby the order of retention is generally consistent with that tabulated by Tomiya (49). The glycome is dominated by typical insect glycans (oligomannosidic, paucimannosidic and hybrid), but also some complex bi-/tri-antennary forms are present; two phosphoethanolamine (PE)-modified glycans in this pool are highlighted in the blue boxes and fucosylated oligomannosidic structures are in light grey boxes. The column was calibrated in terms of glucose units (g.u.). For the core α1,3-fucosylated N-glycans released with PNGase A from larval glycopeptides, refer to Supplementary Figure 1.