*2.2.3 Metal affinity chromatography*

This method exploits the ability of negatively charged sialylated glycans to coordinate with titanium, zirconium or silver [23]. However, metal ion affinity chromatography is not strictly selective for sialylated glycans as negatively charged phosphopeptides or acidic peptides may compete for binding. Additionally, the method does not discriminate between N- and O-linked glycopeptides.

### *2.2.4 Hydrazide chemistry*

Hydrazide chemistry has been widely used for glycosite characterization. Cisdiols within glycans of glycopeptides may be oxidized to aldehydes (using periodate oxidation) forming a non-reversible covalent bond with hydrazide immobilized on a bead. PNGase F is then used to release the formerly N-linked glycosylated peptides to enable N-glycosite determination using MS [24]. Although more commonly used for N-glycosite determination, this chemistry can also be used to enrich glycopeptides having sialylated glycans. In this method, mild periodate treatment selectively oxidizes sialic acids thus enabling capture of sialyated N- and O-glycopeptides on hydrazide beads. The intact glycopeptides can then be selectively released by acid hydrolysis and analyzed by MS [25].

### *2.2.5 Enzyme-mediated O-glycopeptide enrichment*

O-glycopeptides may be enriched using an enzyme-based workflow termed "EXoO" (extraction of O-linked glycopeptides) [26]. This method is enabled by the availability of O-endoproteases (described above). The workflow (**Figure 4**) involves digestion of a protein/biological sample with a standard protease such as trypsin to generate a peptide mixture. The peptides are conjugated to a solid support via the terminal NH2 group on each peptide (*e.g*., Aminolink™ beads, ThermoFisher). An O-endoprotease is used to specifically release O-glycopeptides from the beads. Efficiency of the method is dependent on the specificity of the O-endoprotease. This approach may be practiced with OpeRATOR (Genovis) following chemical modification of sialic acids [18] or with O-glycoprotease (New England Biolabs) which cleaves without pre-treatment to remove or modify sialic acids.
