Does GlycOCATCH bind all O-glycosylated proteins and peptides?

No, GlycOCATCH is designed to bind specifically to mucin type (core 1) O-glycosylated proteins and peptides. The performance is significantly enhanced by removal of sialic acids using SialEXO.

Can OglyZOR be used to deglycosylate peptides generated with OpeRATOR?

Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.

Can I load a digested protein solution directly onto the GlycOCATCH column?

Yes. IMPORTANT! Before you load your sample make sure that the protease in solution is inactivated and that the pH, buffer, chaotrope and NaCl concentrations are within the recommended ranges according to the instructions.

Is it possible to digest the glycoprotein with OpeRATOR first and then enrich the O-glycopeptides on GlycOCATCH?

No, when the glycoprotein is digested with OpeRATOR, the binding capacity of GlycOCATCH is lost.

When and why should I use the OpeRATOR elution procedure?

The OpeRATOR enzyme digests O-glycosylated proteins N-terminally to the serine (S) and threonine (T) glycosylation sites. OpeRATOR therefore facilitates mapping of O-glycosylation sites by LC-MS and as the digested products no longer bind to the GlycOCATCH resin, elution and sample preparation can be achieved in a single step. However, this requires the O-glycosylation sites to be spaced closely together for the OpeRATOR digestion to yield peptides of a size suitable for analysis by LC-MS/MS.

Will OglyZOR work on extended core 1 structures or single HexNAc?

No, OglyZOR recognizes the core 1 disaccharide HexNAcHex (or GalNAcGal) and to some extend also the core 3 disaccharide HexNAc(2) (or GalNAcGlcNAc).

Will OglyZOR leave the N-glycans intact?

OglyZOR only works on desialylated O-glycans therefore is a sialidase (SialEXO) included with the product. SialEXO is not O-glycan specific and will therefore desialylate any N-glycans that are present.

Can I use OglyZOR and PNGaseF in the same reaction?

Yes, we have performed successful experiments with OglyZOR, SialEXO and PNGaseF in the same reaction under native conditions O/N at pH ~7.0.  If denaturing reaction conditions are required for PNGaseF, the reaction with OglyZOR and SialEXO needs to be performed first under native conditions, then add PNGaseF and the required buffer.

Are there any known inhibitors of OpeRATOR?

OpeRATOR is a metalloprotease and as such highly sensitive to chelating agents such as EDTA. Concentrations >5mM lead to complete inhibition of the enzyme. OpeRATOR activity is also moderately inhibited by ZnCl2.

Will OpeRATOR work on simple hexose or pentose?

No, the enzyme recognizes mucin type glycosylation with an inner GalNAc linked to a hexose sugar.

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