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 protein gels was developed. The method employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and purification and in-gel deglycosylation using PNGase F for glycan release. Profiles of the neutral glycans from bovine ribonuclease B, chicken ovalbumin, and human immunoglobulin G (IgG), as well as sialic acid-containing sugars (following esterification of the acidic groups) of bovine fetuin and bovine alpha1-acid glycoprotein, were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and by normal-phase high-performance liquid chromatography following fluorescent labeling. Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS. Between 50 and 100 pmol (1.5 to 15 microg) of glycoprotein applied to the gel was sufficient to characterize its oligosaccharide contents. The 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 database. The technique was used for the characterization of the glycan moieties of human immunodeficiency virus recombinant gp120 (Chinese hamster ovary cells) and to monitor changes in the glycosylation of this glycoprotein 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 with the heavy chains.
Küster
B
B
Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, United Kingdom.
Wheeler
S F
SF
Hunter
A P
AP
Dwek
R A
RA
Harvey
D J
DJ
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000581:Amidohydrolases; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D065089:Glycoside Hydrolase Inhibitors; D006026:Glycoside Hydrolases; D015699:HIV Envelope Protein gp120 / Q000032:analysis; D015497:HIV-1; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000302:isolation & purification; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000520:alpha-Glucosidases
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D065089:Glycoside Hydrolase Inhibitors; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011994:Recombinant Proteins; D017485:1-Deoxynojirimycin; C059896:miglustat; D006026:Glycoside Hydrolases; C042059:glucosidase I; D000520:alpha-Glucosidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase