SEQUENCING OF N-LINKED OLIGOSACCHARIDES DIRECTLY FROM PROTEIN GELS - IN-GEL DEGLYCOSYLATION FOLLOWED BY MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY AND NORMAL-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY/

A generally applicable, rapid, and sensitive method for profiling and sequencing of glycoprotein-associated N-linked oligosaccharides from p rotein gels was developed. The method employed sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation a nd purification and in-gel deglycosylation using PNGase F for glycan r elease. Profiles of the neutral glycans from bovine ribonuclease B, ch icken ovalbumin, and human immunoglobulin G (IgG), as well as sialic a cid-containing sugars (following esterification of the acidic groups) of bovine fetuin and bovine alpha(1)-acid glycoprotein, were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALD I MS) and by normal-phase high-performance liquid chromatography follo wing fluorescent labeling. Oligosaccharides were sequenced using speci fic exoglycosidases, and digestion products were analyzed by MALDI MS. Between 50 and 100 pmol (1.5 to 15 mu g) of glycoprotein applied to t he gel was sufficient to characterize its oligosaccharide contents. Th e identity of all glycoproteins investigated could be confirmed after deglycosylation by in-gel trypsin treatment followed by MALDI MS mass mapping and matching the measured molecular weights to a sequence data base. The technique was used for the characterization of the glycan mo ieties of human immunodeficiency virus recombinant gp120 (Chinese hams ter ovary cells) and to monitor changes in the glycosylation of this g lycoprotein when produced in the presence of a glucosidase I inhibitor . Furthermore, since heavy and light chains of IgG became separated by SDS-PAGE, it could be established that most glycans were associated w ith the heavy chains. (C) 1997 Academic Press.