Structural carbohydrates or glycans cover most cells and secreted proteins. They are implicated in many biological processes, including diseased conditions and biotherapeutics, which necessitate our understanding of glycosylation. However, glycans exhibit incredible structural diversity. Though different approaches have been developed to elucidate the glycan structures, currently there is no single method that can fully characterize glycan structures along with their attachment site on glycoproteins. Though mass spectrometry (MS) has emerged as a popular tool for localizing glycosylation sites it cannot resolve configurational and stereochemical glycan isomers completely. We have established a multi-stage tandem MS workflow that integrates experimentally measured fragment ion ratios to provide detailed glycan structural information. Previous work with protonated monosaccharides (our lab and others) found that tandem MS produces characteristic abundance ratios for the m/z 138 and 144 fragments from GlcNAc and GalNAc. Ratio of 138/(138+144) for both monosaccharides were found to be constant across a wide range of collision energies (CEs). Another ratio of the m/z 84 and 126 ions relative to the sum of all fragments of m/z 204 also behaves in a similar CE independent manner. Moreover, it provides information about the prior linkage configuration. Furthermore, these ion ratios are similar irrespective of the ion trap instrument used (Thermo Orbitrap Fusion vs Thermo LTQ). On the contrary, the same ion ratios obtained using HCD show heavy CE dependence. A series of oligosaccharides varying in monosaccharide composition at the reducing end, non-reducing end, and linkages, for e.g., Gal(ß1-x)GlcNAc, Gal(ß1-x)GalNAc, Neu5Ac(α1-x)Gal(ß1-4)-GlcNAc, where x=2,3,4,6, were examined. A thorough examination of isomeric sialylated oligosaccharides revealed ion ratios from diagnostic ions like m/z 657, 366 that can differentiate linkage isomers, e.g., α2-3 vs. α2-6 linkage and identify their subunit isomers. Thus, we propose the use of these ion ratios as robust metrics for characterizing glycans. The innovation of this approach is in the use of an ion trap mass analyzer that gives reproducible fragmentation propensities that are reliable metrics for interpreting glycan structure. Moreover, this method is implemented on ion traps that are widely used within the glycobiology and proteomics communities, making this workflow compatible with existing technology.

Multistage tandem mass spectrometry for protonated glycan isomer assignment

ACS Fall 2021

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