Journal: Biochimica et biophysica acta. General subjects

Article Title: Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom *

doi: 10.1016/j.bbagen.2019.08.002

Figure Lengend Snippet: 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.

Article Snippet: Thereafter, N-glycans were either released from glycopeptides using peptide:N-glycosidase F (PNGase F, 3 U; Roche) at pH 8 as previously described ( 26 ), with a subsequent digestion of the remaining glycopeptides using PNGase A (recombinant; prepared in house in insect cells) or PNGase Ar (recombinantly produced in Pichia ; New England Biolabs, 15 U) after adjusting to pH 5; the result was a combined PNGase F/A digest as in the case of one larval and one venom preparation.

Techniques: Chromatography, Tandem Mass Spectroscopy, Modification

Journal: Biochimica et biophysica acta. General subjects

Article Title: Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom *

doi: 10.1016/j.bbagen.2019.08.002

Figure Lengend Snippet: 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 Man4-5GlcNAc2-based hybrid glycans nor the PE-, β1,3-Gal and α1·4· alNAc-modified antennae were previously detected in honeybee venom.

Article Snippet: Thereafter, N-glycans were either released from glycopeptides using peptide:N-glycosidase F (PNGase F, 3 U; Roche) at pH 8 as previously described ( 26 ), with a subsequent digestion of the remaining glycopeptides using PNGase A (recombinant; prepared in house in insect cells) or PNGase Ar (recombinantly produced in Pichia ; New England Biolabs, 15 U) after adjusting to pH 5; the result was a combined PNGase F/A digest as in the case of one larval and one venom preparation.

Techniques: Chromatography, Tandem Mass Spectroscopy, Modification

Journal: Biochimica et biophysica acta. General subjects

Article Title: Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom *

doi: 10.1016/j.bbagen.2019.08.002

Figure Lengend Snippet: PNGase F/Ar-released N-glycans were subject to solid phase extraction, whereby the anionic-enriched fraction was eluted with 40% acetonitrile/0.1% trifluoroacetic acid, prior to fluorescent labelling and chromatography on an RP-amide column; each fraction was collected and subject to MALDI-TOF MS. The annotations are based on elution time, MS/MS and digestion data (see Supplementary Figure 6); greyscale structures indicate the elution times of co-fractionating neutral glycans. The column was calibrated in terms of glucose units. Glycans with HexNAc3- or glucuronylated/phosphoethanolamine-modified antennae are highlighted respectively in green and blue boxes.

Article Snippet: Thereafter, N-glycans were either released from glycopeptides using peptide:N-glycosidase F (PNGase F, 3 U; Roche) at pH 8 as previously described ( 26 ), with a subsequent digestion of the remaining glycopeptides using PNGase A (recombinant; prepared in house in insect cells) or PNGase Ar (recombinantly produced in Pichia ; New England Biolabs, 15 U) after adjusting to pH 5; the result was a combined PNGase F/A digest as in the case of one larval and one venom preparation.

Techniques: Chromatography, Tandem Mass Spectroscopy, Modification