In-Gel Enzymatic Release of N-Glycans

Analysis of N-linked glycans, those attached to Asn in an Asn-Xxx-Ser(Thr) motif, where Xxx is any amino acid except proline, requires determination of which consensus sequence is occupied, the extent of occupancy and the structures of the glycans that are attached to each site. Structural analysis of the glycans is most commonly performed following their removal, either chemically (1, 2) or enzymatically, and this chapter describes an enzymatic method for glycan removal using protein N -glycosidase F (PNGase F) (3) from within the SDS-PAGE gels commonly used to separate and purify the glycoproteins. The method is a modification of that described by Küster et al . (4) in 1997. It can be used for mammalian glycans but not for those that are substituted at the 3-position of the reducing terminal GlcNAc residue, commonly found in plants and insects. These glycans are resistant to PNGase F but can be released with PNGase A after trypsinolysis of the protein. Released glycans can then be examined directly by mass spectrometry, usually with MALDI or electrospray ionization, or they can be labeled at their reducing terminus for analysis by chromatographic or electrophoretic techniques. These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.