RE: There are a variety of methods that need to be employed for glycan characterization. If we just think about N-glycans for a moment, which are the most common type of glycosylation we encounter, then one of the most useful techniques is on-line liquid chromatography coupled with mass spectrometric analysis of fluorescently derivatized glycans.
During this analysis at BioPharmaSpec, we enzymically release the glycans from the protein backbone and then tag them with a fluorescent label. We can then separate and detect the
derivatized glycans chromatographically. Since the fluorescent label is in a 1:1 ratio with the glycan, we can use the strength of the fluorescent response to determine the relative abundances of the glycan structures. The nature of what those structures actually are comes from the masses observed as the labelled glycans are analyzed by the mass spectrometer. Structurally related species which have the same mass but different arrangements of monosaccharides can be separated, so profile information can be very detailed.
Data obtained from LC-analysis of 2-AB labeled N-linked oligosaccharides. The N-linked oligosaccharides were released from a monoclonal antibody using PNGase-F.
This is really valuable information, but is just compositional. We also need to know the linkages of the monosaccharides, since this is a key part of assessment of biosimilarity and can have functional significance. We do this through some beautiful chemistry, where we carry out sequential labelling of monosaccharides derived from the glycan species. The labelled monosaccharides are analyzed by gas chromatography mass spectrometry and the fragment ion profiles we see are unique to the individual linkages.
This is my favorite glycan analytical technique – it really is lovely to see the data work so perfectly with the theory. Indeed, you can predict the fragment ions you will see for the different linkages when you know how the chemistry and fragmentation pathways work.
Summed Extracted Ion Chromatogram from GC-MS analysis of a mixture of PMAAs released from a mixture of N-linked oligosaccharides.
FG: So, the use of orthogonality is really important when characterizing glycans?
Yes, orthogonality is really important and we can use a different derivatization and mass spectrometry strategy to generate data orthogonal to the fluorescent profile I just mentioned. This derivatization MS data does not have a chromatographic component so does not allow separation of different isomeric species like the fluorescent data does, but it does give us good supportive mass data and fragment ion information useful for supporting structural assignments. It works with O-glycans too, which is not something that can easily be achieved in the fluorescent labeling sense.
We can also use peptide mapping and identification of glycopeptides to support the information we have on glycan structures, but it should be remembered that in this case glycans can fragment in the source of the mass spectrometer as they ionize. This means we can lose some structural information from larger components and detect some smaller structures due to this fragmentation. For this reason, glycans should be removed from the protein backbone and characterized in their own right, rather than relying on peptide mapping as a mechanism for assessing a glycan population profile.