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February 9, 2026Open Access

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

Key Points

The aim is to develop a protocol for simultaneous profiling of N- and O-glycans from glycoproteins.
Protein reduction and alkylation
N-glycan release and Proteinase K digestion
HILIC enrichment for glycan recovery
N-glycan reduction and permethylation
MALDI-TOF/MS analysis
Successful release and profiling of both N- and O-glycans
Streamlined one-pot protocol improves efficiency
Enhanced sensitivity and resolution of glycans using MALDI-TOF/MS

Abstract

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.

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