Which buffers can be used with the PNGaseF Immobilized column?
The recommended buffer for best performance of the column is TBS buffer at pH 8.6. Other buffers with neutral pH containing 0.15 M NaCl can also be used but optimization may then be required.
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.
Does GalactEXO digest fucosylated glycan structures such as Lewis X or Lewis a?
No. Branching fucose linked to the same residue as the β-galactose inhibits GalactEXO activity and needs to be removed prior to GalactEXO treatment. The FucosEXO enzyme can be used for hydrolysis of α1-2, α1-3 and α1-4 linked fucose.
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.
How long is the reaction time for GalactEXO?
For most substrates such as antibodies or Fc-fusion proteins a 2 h incubation results in hydrolysis of over 95% of the present galactose. More complex substrates with high degree of galactosylation may require longer incubation times.
Does GalactEXO hydrolyze galactose on N and O-glycosylated proteins?
Yes, both beta1-3 linked galactose on O-glycans and beta1-4 galactose on N-glycans are hydrolyzed by GalactEXO.