Monosaccharide and oligosaccharide analysis of isoelectric focusing-separated and blotted granulocyte colony-stimulating factor glycoforms using high-pH anion-exchange chromatography with pulsed amperometric detection.
In this study, a sensitive, straightforward technique is developed for the analysis of glycoprotein O-linked oligosaccharides. Specifically, O-linked oligosaccharides of granulocyte colony-stimulating factor (G-CSF) are analysed by separating charged glycoforms using isoelectric focusing, electroblotting to polyvinylidene difluoride, releasing monosaccharides and oligosaccharide alditols from the blotted glycoprotein bands, and producing chromatographs using high-pH anion-exchange chromatography with pulsed amperometric detection. Using this technique, the O-linked structures of G-CSF produced by recombinant Chinese hamster ovary (CHO) cells are deduced by comparison with monosaccharide and oligosaccharide standards. Lectin blotting and peptide sequencing support the identities of the presumed G-CSF glycoforms. The two major glycoforms determined using this methodology correspond to those determined previously for CHO-produced G-CSF using NMR. Additional glycoforms are also identified in this study, presumably resulting from the presence of N-glycolyneuraminic acid in place of N-acetylneuraminic acid. The utility of this analytical approach is then demonstrated in an analysis of the effect of the extracellular environment on the O-linked glycosylation of G-CSF by recombinant CHO cells. Increasing the level of ammonium ion in the culture medium is shown to reduce the percentage of G-CSF produced with sialic acid linked alpha (2,6) to N-acetylgalactosamine.
Andersen
D C
DC
Department of Chemical Engineering, Stanford University 94305-5025.
Goochee
C F
CF
Cooper
G
G
Weitzhandler
M
M
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012794:Sialic Acids / Q000032:analysis; D013402:Sugar Alcohols / Q000032:analysis / Q000737:chemistry
D009005:Monosaccharides; D009844:Oligosaccharides; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D012794:Sialic Acids; D013402:Sugar Alcohols; D016179:Granulocyte Colony-Stimulating Factor