Enzymatic Release of Glycoprotein N-Glycans and Fluorescent Labeling

Glycosylation, one of the most frequent protein posttranslational modifications, is involved in the mechanisms of cell-cell interactions and immune reactions and is modulated in the course of diseases. In contrary to chemical glycan release, enzymatic cleavage of N-glycans can be performed in any laboratory with relative ease. We present here two robust protocols to achieve N-glycan release. The first one uses trypsin to disrupt protein structure whereas the other involves the use of detergents prior to PNGase F digestion. Thereafter, N-glycans are isolated from peptides using reverse-phase cartridges and are desalted with carbograph cartridges before finally being derivatized with the fluorescent label 2AB.