High-resolution separation of recombinant human interferon-gamma glycoforms by micellar electrokinetic capillary chromatography.
Recombinant human interferon-gamma (IFN-gamma) glycoform populations produced by Chinese hamster ovary cells have been resolved by micellar electrokinetic capillary chromatography (MECC). Separations were performed in uncoated fused silica capillaries at alkaline pH in the presence of micellar concentrations of the anionic detergent sodium dodecyl sulfate (SDS). Maximum resolution was obtained reproducibly with high-ionic-strength borate/SDS electrophoresis buffer. Under the conditions described, glycoform migration time was inversely related to the amount of carbohydrate associated with the protein. Digestion of IFN-gamma with peptide-N-glycosidase F allowed virtual real-time monitoring of glycosidase digests by capillary electrophoresis. Analysis of other digestions with either neuraminidase or endoglycosidase H (endo H) showed most IFN-gamma glycoforms to be sialylated and a minor proportion of glycoforms to be associated with oligomannose structures. While both bovine pancreas ribonuclease B and horse-radish peroxidase glycoforms were separated by this technique, proteins glycosylated at multiple sites such as bovine serum fetuin and human alpha 1-acid glycoprotein were not well resolved by MECC.
James
D C
DC
Research School of Biosciences, Biological Laboratory, University of Kent, Canterbury, United Kingdom.
Freedman
R B
RB
Hoare
M
M
Jenkins
N
N
D000581:Amidohydrolases; D000818:Animals; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods*; D006224:Cricetinae; D004563:Electrochemistry; D006031:Glycosylation; D006596:Hexosaminidases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000302:isolation & purification*; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008823:Micelles; D008832:Microchemistry; D009439:Neuraminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D013997:Time Factors
D008823:Micelles; D011994:Recombinant Proteins; D007371:Interferon-gamma; D006596:Hexosaminidases; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase