Oriented immobilization of PNgase F on a porous polymer monolith for rapid N-glycan release

Abstract

We demonstrate a simple and rapid method for the oriented immobilization of peptide-N4-(Nacetyl- glucosaminyl) asparagine amidase F (PNGase F) on a porous polymer monolith. The oriented immobilization is based on the affinity of glutathione-S-transferase (GST) tagged PNGase F towards glutathione modified monolith prepared in the capillary format. This approach allows the oriented and easily replaceable immobilization of PNGase F for rapid and efficient release of N-linked glycans. The reactor was tested by deglycosylation of several native glycoproteins such as ribonuclease B, fetuin or human immunoglobulin G. The proteins were effectively deglycosylated in several minutes and the released N-linked glycans were analyzed using off-line MALDI/MS or CE/LIF