Rapid Identification of Terminal Sialic Acid Linkage Isomers by Pseudo-MS3 Mass Spectrometry

We used pseudo-triple-stage tandem MS to rapidly differentiate terminal α(2,3)- and α(2,6)-linked sialic acids of glycopeptides and native glycans. The precursor ions generate important diagnostic fragments to distinguish different terminal linked isomeric glycans and glycopeptides based on collision-induced dissociation (CID). The diagnostic ions include hexose linked with N-acetylhexosamine residues, sialic acid with the loss of water, and sialic acid residues. Their values of mass-to-charge ratio (m/z) are at 366.14, 274.09, and 292.10, respectively. The relative intensity ratios (RIR) were calculated by the intensity of signals at m/z 274.09 and 292.10 divided by the intensity of the signal at m/z 366.14. Under optimal conditions of an in-source fragmentation voltage of 70 V and a normalized collision energy of 30 %, the difference in RIR of diagnostic ions between terminal α(2,3)-linked sialic acid of glycopeptide (1.3) and terminal α(2,6)-linked sialic acid of glycopeptide (<0.18) is the largest. Moreover, quantitative calibration curves with different percentages of α(2,3)- and α(2,6)-linked sialoglycopeptides demonstrated that this method applied not only to biantennary N-linked glycan, but also to complex and hybrid N-linked glycans and O-linked glycans. From measurements of the RIR of diagnostic ions, erythropoietin, fetuin, and herceptin have 98, 70, and 100 %, respectively, α(2,3)-linked N-sialic acid of total terminal sugar moieties. N-Glycosylation of the antibody-binding fragment (Fab) moiety of cetuximab has 100 % α(2,6)-linked N-glycolylneuraminic acid terminal glycan linkages. This method can be applied to identify compounds from chemical synthesis. It can also be used for quality evaluation of bio-similar protein drugs.