Separation of human serum transferrin isoforms by high-performance pellicular anion-exchange chromatography.
Glycoproteins are microheterogeneous with respect to their attached oligosaccharides. When these oligosaccharides contain sialic acid, the oligosaccharide microheterogeneity will impart charge heterogeneity to the glycoprotein. We found that two commercial preparations of human serum transferrin (HST), a sialylated glycoprotein, have very different chromatographic profiles when the samples are separated by pellicular anion-exchange chromatography. Each anion-exchange profile contained multiple peaks, which suggested that both glycoproteins have charge heterogeneity. High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) analysis of sialic acids and oligosaccharides released from the two preparations of HST revealed that the two preparations differed in sialic acid and sialylated oligosaccharide content. When oligosaccharides were released from the anion-exchange fractions of the two HST preparations, the HPAEC/PAD oligosaccharide profiles showed that protein retention was directly related to sialylated oligosaccharide content (i.e., the longer a fraction was retained, the greater its sialylated oligosaccharide content). Therefore, the anion-exchange profiles of the two HST preparations are related to their sialylated oligosaccharide content. We believe that pellicular anion-exchange chromatography can be used to quickly monitor gross changes in the sialylation of sialylated glycoproteins due to physiological state, or in the case of recombinant glycoproteins, culture conditions and/or purification.
Rohrer
J S
JS
Dionex Corporation, Sunnyvale, California 94088, USA.
Avdalovic
N
N
D000581:Amidohydrolases / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D014168:Transferrin / Q000737:chemistry / Q000302:isolation & purification*
D006023:Glycoproteins; D009844:Oligosaccharides; D014168:Transferrin; C016735:sialooligosaccharides; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase