FIGURE 3.

NSI-MSⁿ fragmentation of a mixture of two permethylated, isobaric, core 1 trisaccharides bearing glucuronic acid, extracted from Drosophila embryos. Following release from fly embryo powder by reductive (A) or nonreductive (B) β-elimination, glycans were permethylated and characterized by NSI-MSⁿ. Fragmentation of the parent ions corresponding to permethylated HexA₁Hex₁HexNAc-ol (m/z = 752) or permethylated HexA₁Hex₁HexNAc (m/z = 737) demonstrate that both are a mixture of two isobaric O-glycans, one branched and one linear. A, the reduced trisaccharide yields a strong C-ion at m/z = 477, which corresponds to loss of the reducing terminal HexNAc-ol from the linear structure. The Z-ion at m/z = 516 arises from loss of a nonreducing terminal Hex and assigns the HexA residue to a branching position on a minor isomer. B, fragmentation of the nonreduced trisaccharide mixture reveals the same pattern of ions, supporting the predominance of the linear structure, C-ion at m/z = 477, and the presence of a minor branched isomer, Z-ion at m/z = 500 (62, 63). Further fragmentation of the ions at m/z = 477, 500, 504, 516, and 520 by MS³-MS⁴ are consistent with the indicated structures.