The glycome of a cell or tissue is not just N-linked glycans on proteins, although the glycomics literature seems to confer that message. This is due to the availability of analytical tools that N-glycomics analysts have at their disposal (defined sequon, PNGase F, site specific glycopeptide analytical software etc.) and has led to the rapidly developing (N-)glycoproteomics space. However, the “glycome” of a cell or tissue also contains other glycoconjugates such glycosaminoglycans, glycolipids, and non-mucin-type O-glycans (O-mannose, O-fucose, O-glucose etc.). To facilitate method development of the analysis of the “total glycome”, we assembled a small number of commercially available (glyco)proteins that are known to carry various classes of glycans in order to develop a standard glycoprotein mixture to assess the qualitative and quantitative aspects of a total glycomics analysis. Adapting our well-established, PVDF-immobilised glycan release workflow, we added steps to release and analyse other glycan classes from a single sample spot; specifically, to sequentially analyse glycosaminoglycans and glycolipids, as well as protein N-glycans and O-glycans. We applied this approach to the total glycome analysis of mouse brain tissue in a high fat diet model. We thus demonstrate that we are able to carry out a multi-glycomics analysis of different molecular aspects of the glycome from a single sample spot, which will enable a broader view of the changes in the total “glycome” of a cell.