Protocol for simultaneous profiling of N- and O-glycans on glycoproteins using a one-pot format

Here, we present a protocol to simultaneously release and profile N- and O-glycans from glycoproteins in a one-pot format. We describe steps for protein reduction, alkylation, N-glycan release, Proteinase K digestion, HILIC enrichment, N-glycan reduction, N-/O-glycan permethylation while releasing O-glycans, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). For complete details on the use and execution of this protocol, please refer to Ortega-Rodriguez et al.¹.