Figure 1.

Differentiation of N-acetylneuraminic acid (Neu5Ac) linkage isomers using CID fragmentation and subsequent IM-MS analysis in positive ion mode. (A) MS/MS spectra of sialylated N-glycans A2G2S2 (top panel), A3G3S3 (middle panel), and A4G4S4 (bottom panel). Upon CID activation, each sialylated precursor exhibits a characteristic B₃ trisaccharide fragment (m/z 657). (B) Mobilogram of two synthetic trisaccharide standards, which contain a terminal α2,6-linked sialic acid (blue trace) or a terminal α2,3-linked sialic acid (red trace). They can be used as reference to identify the isoforms of the B₃ fragments cleaved from the sialylated glycan precursors. The black dotted line is the original ATD, while the blue and red traces represent the Gaussian fits to indicate α2,6- and α2,3-linked sialic acid isomers. Glycans are represented using the SNFG nomenclature, which depicts monosaccharides as colored symbols.³⁰ The here crucial regiochemistry is defined by the angle of the glycosidic bond.