Analytical Considerations for Effective O- Glycan Characterization
So, there are numerous differences between N-glycan and O-glycan structures but how does this translate into how we analyze them? Are there differences in the techniques we use to study O-glycans compared to those we use for N-glycans? Well, since O-glycans are fundamentally chains of monosaccharides, just as N-glycans are, we can certainly use some of the same techniques to investigate their structures. Just as with N-glycans, we need to release and isolate O-glycans prior to analysis. There is no broad specificity enzyme for the release of O-glycans comparable to the enzyme peptide N-glycosidase F for release of N-glycans. Release of O-glycans is best achieved using the chemical process of reductive elimination followed by purification of the released O-glycans.
Once we have isolated the released O-glycans, we can derivatize (e.g. permethylate) and analyze the derivatives just as we can do for N-glycans, e.g. using Matrix Assisted Laser Desorption Ionization mass spectrometry (MALDI-MS) to generate compositional and fragment ion structural information. MALDI data of permethylated O-glycans can be used to give relative quantitation of the species present. Unlike N-glycans though, O-glycans are not readily amenable to fluorescence analysis by on-line liquid chromatography mass spectrometry, which is a common analytical tool for N-glycans. This is due to the reductive nature of the O-glycan release process not being amenable to the fluorescent labeling chemistry. In practice this is not a major drawback, since O-glycans have limited heterogeneity due to their small size, thus population spread is not significant.
Linkage analysis can also be performed on O-glycans, just as it is performed on N-glycans. It needs to be borne in mind that the GalNAc residue is reduced during the initial release of the O-glycan from the protein and thus, whilst the chemistry will still work, this reduced GalNAc will give rise to unique fragment ions in the mass spectrometric data not found in any monosaccharides derived from N-glycans. It is therefore important to remember that should GalNAc be detected in a monosaccharide analysis experiment, being essentially limited to O-glycans in the therapeutic glycoprotein we are dealing with, it would trigger the need for further O-glycan analytical procedures to be performed for confirmation of the structures.
Identification of sites of O-glycosylation
Identification of site(s) of O-glycosylation within the protein chain can be achieved using peptide mapping, in the same way as we do for N-glycans. The nature of O-glycans does add a further layer of potential complexity in that, if O-glycans are found in clustered regions of the protein, which is quite possible based on the protein structure and functionality of the O-glycans themselves, then mass spectrometric investigations into the O-glycopeptide may not be limited to intact mass information. In these instances, there is a need to generate and assess higher energy O-glycopeptide fragmentation data and use the generated fragment ions to investigate sites of O-glycan attachment within the peptide. Judicious use of proteases can help this by producing different peptides across the region of the O-glycan attachment site(s). This does of course depend on the protein structure and what can reasonably be achieved through proteolysis. Experience in this area is essential to ensure correct experimental design and interpretation of results in what can be a complex series of analyses.
In summary, whilst N-glycans and O-glycans may appear on the surface as simply two different forms of glycosylation, they have developed for specific biological needs and thus have their own unique structures and properties. This means we need to apply glycan analytical techniques that, whilst having some overlap between analysis of the two types, will have specific approaches and considerations based on the differences that exist between these two classes of structure.