Biochemical approaches such as Western blotting may provide supporting evidence that a protein is modified by O-GlcNAc; mass spectrometry (MS) is able to provide definitive evidence as to the presence of O-GlcNAc. Glycoproteomic studies applying MS have contributed to the identification of proteins modified by O-GlcNAc.
As O-GlcNAc is substoichiometric and ion suppression occurs in the presence of unmodified peptides, an enrichment step is usually performed prior to mass spectrometry analysis. This may be accomplished using lectins, antibodies, or chemical tagging. The O-GlcNAc modification is labile under collision-induced fragmentation methods such as collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), so these methods in isolation are not readily applicable for O-GlcNAc site mapping. HCD generates fragment ions characteristic of N-acetylhexosamines that can be used to determine O-GlcNAcylation status. In order to facilitate site mapping with HCD, β-elimination followed by Michael addition with dithiothreitol (BEMAD) may be used to convert the labile O-GlcNAc modification into a more stable mass tag. For BEMAD mapping of O-GlcNAc, the sample must be treated with phosphatatase otherwise other serine/threonine post-translational modifications such as phosphorylation may be detected. Electron-transfer dissociation (ETD) is used for site mapping as ETD causes peptide backbone cleavage while leaving post-translational modifications such as O-GlcNAc intact.
Traditional proteomic studies perform tandem MS on the most abundant species in the full-scan mass spectra, prohibiting full characterization of lower-abundance species. One modern strategy for targeted proteomics uses isotopic labels, e.g., dibromide, to tag O-GlcNAcylated proteins. This method allows for algorithmic detection of low-abundance species, which are then sequenced by tandem MS. Directed tandem MS and targeted glycopeptide assignment allow for identification of O-GlcNAcylated peptide sequences. One example probe consists of a biotin affinity tag, an acid-cleavable silane, an isotopic recoding motif, and an alkyne.  Unambiguous site mapping is possible for peptides with only one serine/threonine residue.
The general procedure for this isotope-targeted glycoproteomics (IsoTaG) method is the following:
# Metabolically label O-GlcNAc to install O-GlcNAz onto proteins
# Use click chemistry to link IsoTaG probe to O-GlcNAz
# Use streptavidin beads to enrich for tagged proteins
# Treat beads with trypsin to release non-modified peptides
# Cleave isotopically recoded glycopeptides from beads using mild acid
# Obtain a full-scan mass spectrum from isotopically recoded glycopeptides
# Apply algorithm to detect unique isotope signature from probe
# Perform tandem MS on the isotopically recoded species to obtain glycopeptide amino acid sequences
# Search protein database for identified sequences
Other methodologies have been developed for quantitative profiling of O-GlcNAc using differential isotopic labeling. Example probes generally consist of a biotin affinity tag, a cleavable linker (acid- or photo-cleavable), a heavy or light isotopic tag, and an alkyne.