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