ANALYTICAL BIOCHEMISTRY 250, 82–101 (1997) ARTICLE NO. AB972199 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 Bernhard Ku ¨ ster, Susan F. Wheeler, Ann P. Hunter, Raymond A. Dwek, and David J. Harvey Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom Received January 31, 1997 fects on protein structure and function. These include A generally applicable, rapid, and sensitive method folding, trafficking, and protein – protein recognition for profiling and sequencing of glycoprotein-associated (1–3). One feature of protein glycosylation is that a N-linked oligosaccharides from protein gels was devel- single site can be occupied by any one of a number of oped. The method employed sodium dodecyl sulfate– glycan structures, and this can give rise to extremely polyacrylamide gel electrophoresis (SDS–PAGE) for heterogeneous glycoprotein populations, termed glyco- protein separation and purification and in-gel deglyco- forms. The glycosylation pattern of a glycoprotein is not sylation using PNGase F for glycan release. Profiles of random but dependent on the cell type, physiological the neutral glycans from bovine ribonuclease B, chicken conditions, and, in some cases, disease states. ovalbumin, and human immunoglobulin G (IgG), as well Traditional methods for structural investigation of as sialic acid-containing sugars (following esterification protein-bound oligosaccharides employ either chemical of the acidic groups) of bovine fetuin and bovine a 1 -acid (4, 5) (e.g., hydrazinolysis) or enzymatic (6) (e.g., glycoprotein, were obtained by matrix-assisted laser de- PNGase F) 1 release of the glycan moiety from the glyco- sorption/ionization mass spectrometry (MALDI MS) and protein followed by separation of the mixture into its by normal-phase high-performance liquid chromatogra- phy following fluorescent labeling. Oligosaccharides were sequenced using specific exoglycosidases, and di- 1 Abbreviations used: a-CN, a-cyano-4-hydroxycinnamic acid; gestion products were analyzed by MALDI MS. Between AGP, a 1 -acid glycoprotein; Asn, asparagine; Asp, aspartic acid; 2- 50 and 100 pmol (1.5 to 15 mg) of glycoprotein applied to AB, 2-aminobenzamide; APS, ammonium peroxodisulfate; CHO the gel was sufficient to characterize its oligosaccharide cells, Chinese hamster ovary cells; DHB, 2,5-dihydroxybenzoic acid; contents. The identity of all glycoproteins investigated DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EMBL, European Molecular Biology Laboratory; Endo-H, endo-b-N-acetylglucosamini- could be confirmed after deglycosylation by in-gel tryp- dase H; Fuc, F, L-fucose; FWHM, full width at half-maximum; Gal, D- sin treatment followed by MALDI MS mass mapping and galactose; Glc, glucose; GlcNAc, N-acetyl-D-glucosamine; GU, glucose matching the measured molecular weights to a se- units; H, hexose; HIV, human immunodeficiency virus; HPAEC/PAD, quence database. The technique was used for the char- high-performance anion-exchange chromatography with pulsed amp- acterization of the glycan moieties of human immunode- erometric detection; HPLC, high-performance liquid chromatogra- ficiency virus recombinant gp120 (Chinese hamster phy; IgG, human immunoglobulin G; MALDI MS, matrix-assisted ovary cells) and to monitor changes in the glycosylation laser desorption/ionization mass spectrometry; Man, D-mannose; N, N-acetylhexosamine; NB-DNJ, N-butyldeoxynojirimycin; NMR, nu- of this glycoprotein when produced in the presence of clear magnetic resonance; NP-40, Nonidet P-40, octylphenoxypolye- a glucosidase I inhibitor. Furthermore, since heavy and thoxyethanol; NP-HPLC, normal-phase high-performance liquid light chains of IgG became separated by SDS– PAGE, it chromatography; Ova, chicken ovalbumin; PEG, polyethyleneglycol; could be established that most glycans were associated PNGase F, peptide N 4 -(acetyl-b-glucosaminyl)asparagine aminidase with the heavy chains.  1997 Academic Press F; RNase B, bovine pancreatic ribonuclease B; RT, room tempera- ture; S, sia, sialic acid (N-acetylneuraminic acid); SDS – PAGE, so- dium dodecyl sulfate – polyacrylamide gel electrophoresis; TEMED, Glycosylation is a common co- and posttranslational tetramethylethylenediamine; TFA, trifluoroacetic acid; TOF, time-of- flight; Tris, tris(hydroxymethyl)aminomethane; w/w, weight/weight. modification of proteins which may have profound ef- 82 0003-2697/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.