Analysis of N-linked oligosaccharides released from glycoproteins separated by two-dimensional gel electrophoresis

Protocols have been developed for the characterization of carbohydrate cova lently attached (N-linked) to an asparagine residue in glycoproteins, after separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE ). Mixtures of proteins teach at a level from 0.5 to 50 mu g) were resolved in the first dimension according to their isoelectric points (pI, followed by separation in the orthogonal axis on the basis of their molecular weigh ts. Glycans were released directly from excised gel spots after digestion w ith PNGase F, with or without prior treatment with trypsin. In a third meth od, glycoproteins were electroblotted onto poly(vinylidene difluoride) befo re glycans were released by PNGase F. For all these procedures profiles of the neutral and sialic acid-containing oligosaccharide mixtures were obtain ed after derivatization with 3-acetamido-6-aminoacridine, and analysis by m atrix-assisted laser desorption/ionization time-of-flight mass spectrometry and/or high-performance liquid chromatography. Potential applications to p roteomics are discussed. (C) 2000 Academic Press.