Fig. 6.

Example of MS/MS and digestion data for PNGase Ar-released N-glycans from Caenorhabditis elegans L4 larvae. RP-HPLC fractionated glycans were subject to MALDI-TOF MS and MS/MS in positive mode before and after chemical or enzymatic treatment. The MS/MS spectra are annotated with the fraction name (L4liqAr or L4plaAr and the retention time), the m/z value, key fragments and summarized or shown sensitivity to α- or β-galactosidase, α-mannosidase, α1,2-fucosidase, or hydrofluoric acid, resulting in the indicated losses of fucose (F), methylated fucose (FMe), or hexose (H). A–J, comparison of five isomers of m/z 1281 (Hex₃HexNAc₂Fuc₂) and two isomers of m/z 1443 (Hex₄HexNAc₂Fuc₂) with indications of differences in core Y1 fragments and effects of enzymatic or chemical treatments. K–M, comparison of two isomers of m/z 1589 (Hex₄HexNAc₂Fuc₃) found in the L4 liquid glycome and the effect of HF treatment of one of them resulting in replacement of the proximal Y₁ difucosylated fragment at m/z 592 with a monofucosylated one at m/z 446 in addition to loss of the distal GalFuc motif. N–T, comparison of five isomers of m/z 1589 in the L4 plate glycome and the effect of α1,2-fucosidase on two of them; the summed evidence shows variations in the positions of the fucose and galactose residues. The occurrence of difucosylated and trifucosylated chitobiose cores is in accordance with the defined activities of C. elegans FUT-1, FUT-6, and FUT-8 (14), the absence of such cores from the fut-1;fut-6;fut-8 triple knockout strain (12), and others’ ESI–MSⁿ data on permethylated C. elegans N-glycans (21, 25). U and V, depiction of the separation of Hex₃HexNAc₂Fuc₂ and Hex₄HexNAc₂Fuc₃ isomers by RP-HPLC; the overlaid liquid and plate chromatograms in Figure 5 are shown in red and black, respectively. ESI, electrospray ionization; RP, reverse phase.