FIGURE 4.

NSI-MSⁿ fragmentation of a permethylated, glucuronylated O-Fuc glycan 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 HexA₁HexNAc₁deoxy-Hex-ol (m/z = 722) or permethylated HexA₁HexNAc₁deoxy-Hex (m/z = 706) reveal a branched trisaccharide with a reducing terminal Fuc. A, fragmentation of the reduced trisaccharide yields strong Z-ions at m/z = 445 and 472, which correspond to loss of nonreducing terminal HexNAc and HexA branches, respectively. The major MS/MS ion at m/z = 576 arises from an incompletely defined cross-ring cleavage through the HexA residue (broken line arrow). The same cross-ring cleavage is evident as a very minor fragment in MS² of the glucuronylated core 1 disaccharide (see Fig. 3) in both reduced (m/z = 606) and nonreduced (m/z = 593) forms, suggesting that glycosidic linkage to a deoxy-Hex imparts stability. Although the cleavage and possible rearrangements associated with this cross-ring event are not resolved, the resulting X-ion fragments in MS³ to produce the expected losses for the predicted structure: a Z-ion at m/z = 472 from loss of the residual HexA, a ^0,4X-ion at m/z = 398 from cross-ring cleavage through the HexNAc, and a C-ion at m/z = 298 from liberation of the nonreducing terminal HexNAc branch. B, fragmentation of the nonreduced trisaccharide reveals the same pattern of supporting ions as well as the Y-ion at m/z = 301 in MS³, corresponding to the reducing terminal deoxy-Hex. Further fragmentation (MS⁴-MS⁵) identified substitution positions for the HexA and HexNAc termini (supplemental Fig. 6).