Glycosylation is well known to alter and fine-tune the functions of glycoproteins. A wide range of biological functions has been described such as conferring proteolytic resistance and mediating or inhibiting inter- and intracellular interactions [1]. As a matter of fact, protein glycosylation does not introduce a single, universally definable functionality. Thus detailed knowledge on the protein specific glycosylation and its site distribution is crucial for any systematic investigation of the functional aspects that protein glycosylation is playing on individual glycoproteins.

In that context mass spectrometry has become the method of choice for the identification and in depth primary structure characterisation of glycoconjugates. Nevertheless, it lacks the intrinsic ability to easily distinguish structural isomers of isobaric compounds, and the non-template based bio-synthesis of glycans as well as their non-linear structures pose additional challenges in glycan structure assignment by MS.

This presentation will give an overview on the peculiarities of protein glycosylation analysis and will focus in particular on advantages, challenges and limitations for in-depth glycoconjugate characterisation by MS. Synthetic glycopeptides, where both the peptide and glycan moieties can be adjusted, have become essentials tools for the systematic investigation, benchmarking and development of novel approaches for qualitative and quantitative glycopeptide analysis [2, 3]. In combination with orthogonal approaches for the analysis of the released N-glycans (porous graphitized carbon nanoLC ESI-MS/MS & CGE-LIF) and O-glycans (PGC nanoLC ESI-MS/MS) [4, 5], a detailed glycomics and glycoproteomics map of the entire human Immunoglobulome (IgA, sIgA, IgD, IgE, IgG and IgM) has been established, revealing the potential type II receptor sites present on human Immunoglobulins.