SSSMuG: Same Sample Sequential Multi-Glycomics

The mammalian glycome is structurally complex and diverse, composed of many glycan classes such as N- and O-linked glycans, glycosaminoglycans (GAGs), glycosphingolipids (GSLs) and other distinct glycan features such as polysialic acids (PolySia), sulfation and proteoglycan attachment stubs. Various methods are used to analyse these different components of the glycome, but they require pre-fractionated/partitioned samples to target each glycan class individually. To address this need for a knowledge of the relationship between the different glycan components of a biological system, we have developed a sequential release workflow for analysis of multiple conjugated glycan classes (PolySia, GAGs, GSL glycans, N-glycans O-glycans) from the same tissue lysate, termed SSSMuG: Same Sample Sequential Multi-Glycomics. With this sequential glycan release approach, five glycan classes were characterized (or four glycan classes plus proteomics) from a single sample using enzymatic or chemical release from the same spot immobilized on a polyvinylidene difluoride membrane. The various released glycan classes were then analysed by HPLC and/or MS techniques. Compared to single glycan class release approaches, SSSMuG is able to identify more glycans and more proteins with higher intensity analytical peaks and provides a better comparative normalisation of the different glycan classes of the complex glycome.