Fig. 7.

Negative mode LC-ESI–MSⁿ of a glucuronylated glycan. a The Hex₃HexNAc₅Fuc₁HexA₁ m/z 1920 glycan eluting at 14 g.u. (see also corresponding positive and negative mode MALDI-TOF–MS/MS data in b and c, whereby the latter shows some of the same non-cross-ring fragments as the ESI-MS/MS) and containing a HexA1-4HexNAc-4HexNAc-4GlcNAc motif as shown by MS² of ions at m/z 958.64 (Hex₃HexNAc₅HexA₁Fuc₁-PA, [M−2H]²⁻) and MS³ of ions at m/z 784.20 (HexNAc₃HexA₁, [M−H]⁻) and at m/z 581.20 (HexNAc₂HexA₁, [M−H]⁻). Fragmentation ions at m/z 664 and 1390 were annotated as ^2,4A and ^0,2A cleavage of penultimate GlcNAc, both cleavages are diagnostic ions for N-glycans⁶⁹. The presence of fragmentation ions at m/z 1036 (^0,3A of βMan) and “D ions” at m/z 1090/1108 suggest the HexNAc₃HexA₁ motif linked to Man on the 6-antenna. No fragments are compatible with the presence of terminal HexNAc, while a series of B ions at m/z 378, 581 and 784 suggests a linear HexA-HexNAc-HexNAc linked to β1,2-linked GlcNAc on the 6-antenna. To determine the linkage between HexA-HexNAc-HexNAc, MS³ of the fragmentation ions at m/z 581, which contain HexNAc₂HexA₁, was performed. Fragments at m/z 295/277 (^0,2A_HexNAc/^0,2A_HexNAc-H₂O) suggest terminal HexA linked to C₄ of HexNAc; the ions at m/z 295 would be compatible with either a 3-, 4- or 6-linkage, but the m/z 277 fragment is most compatible with a 4-linkage. In addition, fragmentation ions at m/z 498/480 (^0,2A_HexNAc/^0,2A_HexNAc-H₂O) indicate the presence of HexA-4HexNAc linked to C4 of HexNAc. Taken together, this special N-glycan antennal motif was annotated as a HexA-4HexNAc-4HexNAc sequence linked to the β1,2-linked GlcNAc on the 6-antenna