Sialic Acid Linkage Identification in Glycosylated Peptides Using Multitandem Collision-Induced and Electronically Excited Dissociation Mass Spectrometry

In our previous publication [<i>Anal. Chem.</i> <b>2023</b>, <i>95</i>, 7458-7467], we demonstrated the use of electron capture dissociation for Sa linkage analysis in N-glycans; however, its application was challenging because its diagnostic capabilities depended on the size of the glycans and peptide backbones. In this work, we propose a universal method for analyzing Sa linkages in N-glycans and O-glycans that is applicable to both glycopeptides and free glycans. Specifically, electronically excited dissociation (EED) is applied to the antennary SaGalHexNAc fragments that collision-induced dissociation (CID) produces from glycopeptides and glycoproteins. We found that EED on the fragments produced the Z₂^• fragment when Sa was linked in α(2,3) in N-glycans, but it was negligibly weak in the cases with α(2,6) linkages. This method was expanded to O-glycans, though the discrimination between the two Sa linkages was not as prominent as that of N-glycans. We introduced four synthesized authentic glycan standards: SaGalHexNAc-OH in the free glycan forms in two Sa-Gal linkages and HexNAc: GlcNAc and GalNAc established the calibration of the Sa-linkage diagnostics in both sialylated N- and O-glycans. We confirmed that the water-lost products by CID from the free form standards have the same structures as the CID products from the native glycans and glycopeptides, which is followed by EED. The established methods were successfully applied to native glycopeptides and glycoproteins, including egg yolk glycopeptide, bovine fetuin, and human erythropoietin.