Reproducible and sensitive determination of charged oligosaccharides from haptoglobin by PNGase F digestion and HPAEC/PAD analysis: glycan composition varies with disease

Many studies have reported changes in the carbohydrate structure of serum glycoproteins in disease, but this information is often of limited value for understanding disease mechanisms because it is obtained with simple and/or indirect methodologies that determine only one structural feature. On the other hand, more detailed carbohydrate methodologies are time-consuming and require a lot of purified material. Using haptoglobin (Hp) as a model protein, a new procedure was devised that determined the oligosaccharide composition of very small amounts of Hp in a relatively short time. The Hp was purified by batch affinity-chromatography, oligosaccharides were removed with PNGase F, and the oligosaccharide composition of charged species was determined using HPAEC/PAD (Dionex carbohydrate analyser). The method was applied to the analysis of Hp from eight healthy individuals and 37 patients with different inflammatory diseases or cancers. Twenty-seven oligosaccharides were consistently detected, but the majority could not be identified. However, by calculating retention times relative to the sialylated biantennary peak (Neu5Ac2-3/6Gal1-4GlcNAc1-2Man1-6(Neu5Ac2-3/6 Gal1-4GlcNAc1-2Man1-3)Man1-4GlcNAc1-4GlcNAc) it was possible to compare profiles quantitatively. Although no peak was identified as disease-specific, characteristic and reproducible profiles were obtained. Particularly striking were reductions in the major peaks in Crohn's disease, rheumatoid arthritis, stomach cancer, accompanied by increases in unidentified peaks. Previous studies suggested that many of the unknown peaks were due to increased sialylation and fucosylation. Only small changes in patterns were observed for breast and ovarian cancer. The new procedure will be very useful in the characterization of oligosaccharide composition of glycoproteins in clinical specimens.