Fig. 2. St8sia3 depletion impaired the addition of terminal sialylated units to striatal N-glycans.

Permethylated N-glycans derived from WT (+/+) and St8sia3-KO (−/−) striatal tissues were subjected to a nanoLC-MS²-product-dependent MS³ analysis, in which the MS² fragment ions representing NeuAc₂⁺ (m/z 737) and NeuAc₂-Hex-HexNAc⁺ (m/z 1186) were additionally selected for MS³ analyses upon detection. a The summed intensity of a panel of diagnostic MS² ions allowed the rapid assessment of the relative abundance of each glycotope at the glycomic level, as annotated by cartoon symbols and respective m/z values indicated on the x-axis. The magnified panel showed a significant reduction in the level of the m/z 737 ion in St8sia3-KO mice. b The summed intensity of the diagnostic MS³ ions that verified the NeuAc-NeuAc disialyl unit, i.e., m/z 376 from 737 and m/z 737 from 1186, indicated a drastic reduction in St8sia3-KO mice, whereas the ions that identified a type 1 chain sialylated at 2 distinct positions, i.e., m/z 589 from 1186, actually increased. The cartoon drawings illustrate how these diagnostic MS³ ions validated and added confidence to the identification of the isomeric disialylated glycotopes. c A search of the LC-MS²/MS³ data for presence of terminal trisialylated LacNAc using the diagnostic ions at m/z 1547 led to the identification of multisialylated N-glycans in the WT samples, but not the St8sia3-KO samples, and these antennary glycotopes were observed at low level, along with other permutations of mono- and disialylated antennary glycotopes. The inset shows the correct monoisotopic mass for the selected precursor ion, while the HCD MS² spectrum contained the complement of oxonium ions, supporting the assigned glycosyl composition, including an ion at m/z 1186 that was further selected for MS³ and yielded a product at m/z 737. Glycomic mapping of striatal N-glycans revealed a significant reduction in the number of terminal disialyl units in St8sia3-KO mice. n = 3 for each genotype with duplicates. *P < 0.05, **P < 0.01 and ***P < 0.001, as compared with WT control (two-tailed unpaired Student’s t-test). All values are presented as the means ± S.E.M