Although the analysis of glycosylation patterns has already been established for the identification of biomarkers and monitoring of therapeutic glycoproteins, localization of the glycosylation sites and determination of amino acid sequences are also required for complete glycopeptide characterization.
Currently, the analysis of glycopeptides is still a challenge. The generally lower ionization efficiency in comparison to non-glycosylated peptides and a high micro-heterogeneity (different glycan structures attached to a single glycosylation site) makes the analysis of N- and O-glycopeptides difficult. Collision Induced Dissociation (CID) of glycopeptides mainly yields glycan fragments but produces limited information about the peptide backbone. In contrast, Electron Transfer Dissociation (ETD) cleaves peptides N-Cα bonds, whereas the glycan remains attached to the amino acid residue.
Therefore, the combination of CID and ETD fragmentation represents an excellent tool for the comprehensive characterization of glycopeptides. Typically, ETD fragmentation of glycopeptides suffers from low charge states in combination with their high m/z. In order to significantly improve ETD efficiency, the new CaptiveSpray nanoBooster ionization source (utilising a solvent-enriched sheath gas) has been used to enhance glycopeptides charge states on the one hand and increase their overall signal intensities on the other hand. The strongest influence is observed for glycopeptides with large glycans attached and/or high degrees of sialylation. Based on this methodology, this study demonstrates the successful application of the different fragmentation techniques – CID and ETD – for N- and O-glycopeptides.