Technical Director for Structural Analysis
Dr. Richard Easton is the Technical Director for Structural Analysis at BioPharmaSpec, an independent analytical CRO with labs in the UK & US. He has over 20 years’ experience in glycoprotein structural characterization using mass spectrometry and is well acquainted with the various regulatory requirements for biopharmaceutical development.
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.