Glycoconjugates are increasingly used as anti-bacterial vaccines. In their simplest forms, they are similar to glycoproteins. However, even the complete characterization of glycoproteins and glycopeptides continues to present difficulties. While mass spectrometry can provide the mass of the molecule of interest and site-specific information for the glycosylation pattern, it is difficult to quantify glycoforms in the same sample. Another difficulty is with isobaric material as the compositional information (stereochemistry) is lost. NMR has proven to be useful in not only filling in these gaps, but also in providing a significant amount of information from a single sample. Using the HSQC-TOCSY provided the ability to observe glycans on full length protein with the beneficial consequence of eliminating protein resonances. This relatively common NMR method efficiently reduces spectral overlap caused by protein resonances crowding and overlapping with the carbohydrate peaks in the same spectral region, thus facilitating characterization. The method works well on natural abundance 13C samples of reasonable concentrations (>5 mg/mL) and experiment time can be further reduced by the use of NUS. This opens the door to the thorough characterization of glycans in the context of very large proteins which would otherwise be very difficult. This is one step towards characterizing glycoconjugates and larger conjugate vaccines. By combining these NMR methods with mass spectrometry experiments, sites of oligosaccharide attachment, the ratio of different oligosaccharides, and potentially the amount of oligosaccharide can be determined for glycopeptide vaccine mimics.
