The mammalian glycome is structurally complex and diverse, composed of many glycan classes such as protein N- and O-linked glycans, glycosaminoglycans (GAGs), glycosphingolipids (GSLs) and other distinct glycan features such as polysialic acids (PolySia), sulfation and proteoglycan attachments. Various methods are used to analyze 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 structural 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, plus proteomics, of a single brain lysate sample, using enzymatic and/or chemical release from a sample immobilized on a polyvinylidene difluoride membrane. The various released glycan classes were then analyzed by HPLC and/or MS techniques using different commonly used analytical setups. Compared to single release approaches, SSSMuG was able to identify more glycans and more proteins with higher intensity analytical peaks than individual analyses, and provided a better comparative normalisation of the different glycan classes of the complex glycome and proteome. To this end, the SSSMuG technology workflow will be a foundation for a paradigm shift in the field, transforming glyco-analytics and facilitating the push towards tissue multi-glycomics, together with improved proteomics, of the very same sample.