Fig. 6
From: Highly modified and immunoactive N-glycans of the canine heartworm
Example MS/MS data of glucuronylated N-glycans. a–c MALDI-TOF–MS/MS (with MS in insets) of the 2D-HPLC purified m/z 1918 (negative) or 1920 (positive) glycan eluting at 8.2 g.u. (Fig. 5b) before (a, b) and after (c) Helix pomatia β-glucuronidase treatment; removal of the glucuronic acid residue correlates with loss of 176 Da from the parent and of the positive mode B-fragment ion at m/z 583 (HexA1HexNAc2). In general, the positive ion mode MS/MS spectra of the protonated forms of glucuronylated glycans were more intense and more informative than the negative-ion mode MS/MS spectra, despite the excellent ionisation in negative mode of the parent species. d–i Positive mode MS/MS of monoglucuronylated N-glycans, including two isomeric forms of m/z 1920 differing in the position of the fucose residue (as shown by the m/z 932 HexA1HexNAc3Fuc1 fragment for one form), two hybrid, one core di-fucosylated and one phosphorylcholine-modified structures found in RP-amide fractions derived from HIAX anionic pools b, c and h; the presence of an upper arm antenna on the di-fucosylated glycan (h) is compatible with the requirement of nematode core α1,3-fucosyltransferase for an unsubstituted α1,3-mannose residue, whereas the m/z 592 Y-fragment defines the core difucose modification (see also Supplementary Figure 5B and C). j–p Positive ion mode MS/MS of 2D-HPLC-purified di-, tri- and tetra-glucuronylated N-glycans (see Fig. 5h, j–l). Losses of one, two or three HexA1HexNAc2-3 motifs (Δm/z 582 or 785) are indicated in addition to selected antennal B- and pyridylamino-containing Y-fragment ions. The concluded structures reflect the presence of corresponding non-glucuronylated tri- and tetra-antennary forms in the neutral pools with 4-linked GlcNAc leading to later RP-amide elution than a 6-linked GlcNAc. The two m/z 2502 glycans (j, k) differ in the location of the fucose residue, while the m/z 3521 glycan (o) elutes earlier than that of m/z 3667 (p) due to the retentive effect of the core α1,6-fucose; for further characterisation of bi-antennary forms including glycosidase digests, refer to Fig. 8 and Supplementary Figures 13–15
