Enhanced glycan nanoprofiling by weak anion exchange preparative chromatography, mild acid desialylation, and nanoliquid chromatography-mass spectrometry with nanofluorescence detection.

The structural characterization and quantification of the glycome of cells and glycoproteins is necessary for the understanding of glycan functions in Biology, the development of diagnostics tests, and the monitoring of glycoprotein pharmaceuticals. Classical N-glycan characterization methods involve enzymatic release followed by derivatization with a fluorochrome and separation by normal-phase HPLC. We have recently developed glycan nanoprofiling, a method for the simultaneous quantification and characterization of the N-glycans without the need of external standardization. Although glycan nanoprofiling allows the characterization of both neutral and sialylated glycans within the same chromatographic run, a significant drawback is the coelution of similar glycans when complex glycan mixtures are analyzed. To overcome this problem, we have developed enhanced glycan nanoprofiling. This new method introduces a weak anion-exchange HPLC separation step to fractionate glycans according to their sialic acid content followed by a mild acid desialylation. Glycans are then resolved by nano-LC-coupled ESI-MS with an intercalated nanofluorescence detector. Neutral glycans have a better analytical separation, better ionization profiles, and provide significantly higher MS signals allowing a detailed characterization of rare glycan species. Enhanced glycan nanoprofiling is a powerful approach that provides a fast and sensitive alternative to available N-glycan profiling methods.