Rapid deglycosylation of proteins for carbohydrate and protein characterization by mass spectrometry using Endo H-bound magnetic beads

摘要

Endo H is a good glycosidase to use for glycoproteomics. The enzyme is active both at low pH and with buffers that won't suppress MS signal. Additionally, the reaction of Endo H leaves a GIcNAc scar on the protein thereby making glycosylation sites identifiable by MS/MS analyses. The activity of Endo H is affected by the protein substrate: the deglycosylation of ovalbumin was incomplete even after an overnight incubation whereas RNase B was completely deglycosylated after 30 min. With the Endo H-bound Magnetic Beads both intact protein and deglycosylated sugar analyses can be conducted from the same sample without any additional sample preparation. In contrast, Endo H in-solution poses a problem because the enzyme can co-elute with the deglycosylated substrate when using a "step-gradient". Additionally, to identify sugars resulting from deglycosylation with Endo H in-solution, further clean up using Carbon Graphite Zip Tips is required (data not shown).