A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format

Glycosylation, including N-glycosylation and O-glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein-based drug product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well-established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by Proteinase K, selective N-glycan reduction, and O-glycan release by β-elimination during permethylation of both N- and O- glycans. Glycan structural assignments, and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is an accurate and reproducible approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.