김아빈 (충남대학교)

안현주 (충남대학교)

김아빈 (충남대학교)

YINDONGTAN (충남대학교)

오명진 (충남대학교)

안현주 (충남대학교)

Gangliosides are sialic acid-containing glycosphingolipids (GSLs) abundant in neural cell membranes and play important roles in neural development and signaling. Although lipid C=C positions influence enzyme selectivity, analysis of C=C positional isomers remains limited. Here, we characterized ganglioside C=C positional isomers using LC/oxygen attachment dissociation (OAD)-Q-TOF MS/MS. Radical-driven OAD generated C=C position-specific diagnostic ions for LacCer, GM1, GD1a, GM2, and GM3 standards, and integration with LC/CID-MS/MS enabled structural characterization of intact GSLs. For LacCer, a ganglioside biosynthetic precursor, CID provided only the composition LacCer d18:1/24:1, whereas OAD enabled assignment of the C=C positions as d18:1(Δ4)/24:1(n-9) based on diagnostic ions, including m/z 875.595 (n-8_n-9 cleavage), 834.594 (n-10_n-11 cleavage), and 762.499 (Δ4 cleavage). Interestingly, the 210.234 Da neutral loss from Δ4 cleavage was also observed in GM1, GD1a, GM2, and GM3, confirming that their sphingoid bases were d18:1(Δ4). Despite being derived from ovine, porcine, and bovine sources, all four standards exhibited the same Δ4 C=C position. Furthermore, the applicability of this approach was demonstrated using GSLs extracted from whole mouse brain. It enabled precise structural characterization of intact gangliosides, including C=C positional assignment, and may facilitate detailed structural analysis of GSLs in biological samples.