Fig. 3
From: Highly modified and immunoactive N-glycans of the canine heartworm
Analysis of example phosphorylcholine and fucose-modified N-glycans. a–f RP-amide HPLC, MALDI-TOF–MS and MS/MS analysis of an example 2D-isolated fraction before (a, c, e) and after (b, d, f) hydrofluoric acid treatment. A shift to higher elution due to altered hydrophobicity of the glycans (b) and alterations in the MS/MS spectra (removal of one fucose and one phosphorylcholine from m/z 1852 or of two fucoses from m/z 2239 correlating with losses of the m/z 184 (PC + H2O), 369 (GlcNAc1PC), 718 (HexNAc2Fuc1PC) and 902 (HexNAc3Fuc2) B-fragment ions) verify the proposed compositions, whereas the ability to remove one GalNAc from both with HEX-4 (data not shown) enabled the proposition of the isomeric structure. g–l MS/MS of other phosphorylcholine-containing glycans as well as one example (j) after hydrofluoric acid treatment; other sensitivities to this reagent or to hexosaminidases are noted. The contrast in the spectra of the two isomers of Hex3HexNAc4Fuc1PC1 (m/z 1706; g, h) is due to the position of the fucose residue. Selected B- and Y-fragments are annotated as well as certain losses of fucose or hexose; due to the excellent ionisation of PC-containing fragments (e.g. at m/z 718 and 572), the presence of core fucose is shown by the loss of 445 Da from the parent and by the presence of an m/z 446 Y-fragment observable after hydrofluoric acid treatment. The terminal position of phosphorylcholine is inferred by the minimal fragment containing both the PC and a hexose (i.e. m/z 734 [Hex1HexNAc2PC1], rather than m/z 531 [Hex1HexNAc1PC1] as in other nematodes). See Fig. 4 and Supplementary Figure 9 for further examples of data on phosphorylcholine-modified N-glycans
