Collisionally activated dissociation and electron capture dissociation provide complementary structural information for branched permethylated oligosaccharides

Doubly charged sodiated and permethylated linear malto-oligosaccharides ({Glc}(6)-{Glc}(9)), branched N-linked glycans (high-mannose type GlcNAC(2)Man(5-9), and complex asialo- and disialylated-biantennary glycans) were analyzed by tandem mass spectrometry using collisionally-activated dissociation (CAD) and hot electron capture dissociation (ECD) available in a custom-built ESI FTICR mass spectrometer. For linear permethylated malto-oligosaccharides, both CAD and hot ECD produced glycosidic cleavages (B, Y, C, and Z ions), cross-ring cleavages (A- and X-type), and internal cleavages (B/Y and C/Y type) to provide sequence and linkage information. For the branched N-linked glycans, CAD and hot ECD provided complementary structural information. CAD generated abundant B and Y fragment ions by glycosidic cleavages, whereas hot ECD produced dominant C and Z ions. A-type cross-ring cleavages were present in CAD spectra. Complementary A- and X-type cross-ring fragmentation pairs were generated by hot ECD, and these delineated the branching patterns and linkage positions. For example, (0,4)A(n) and (3,5)A(n) ions defined the linkage position of the major branch as the 6-position of the central core mannose residue. The internal fragments observed in CAD were more numerous and abundant than in hot ECD spectra. Since the triply charged (sodiated) molecular ion of the permethylated disialylated-biantennary N-linked glycan has relatively high abundance, it was isolated and fragmented in a hot ECD experiment and extensive fragment ions (glycosidic and complementary pairs of cross-ring cleavages) were generated to fully confirm the sequence, branching, and linkage assignments for this glycan.