REPRODUCIBLE AND SENSITIVE DETERMINATION OF CHARGED OLIGOSACCHARIDES FROM HAPTOGLOBIN BY PNGASE-F DIGESTION AND HPAEC PAD ANALYSIS - GLYCANCOMPOSITION VARIES WITH DISEASE/

Many studies have reported changes in the carbohydrate structure of se rum glycoproteins in disease, but this information is often of limited value for understanding disease mechanisms because it is obtained wit h simple and/or indirect methodologies that determine only one structu ral feature. On the other hand, more detailed carbohydrate methodologi es are time-consuming and require a lot of purified material. Using ha ptoglobin (Hp) as a model protein, a new procedure was devised that de termined the oligosaccharide composition of very small amounts of Hp i n a relatively short time. The Hp was purified by batch affinity-chrom atography, oligosaccharides were removed with PNGase F, and the oligos accharide composition of charged species was determined using HPAEC/PA D (Dionex carbohydrate analyser). The method was applied to the analys is of Hp from eight: healthy individuals and 37 patients with differen t inflammatory diseases or cancers. Twenty-seven oligosaccharides were consistently detected, but the majority could not be identified. Howe ver, by calculating retention times relative to the sialylated bianten nary peak (Neu5Ac alpha 2-3/6Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6( Neu5Ac alpha 2-3/6Gal beta 1- 4GlcNAc beta 1-2Man alpha 1-3)Man beta 1 -4GlcNAc beta 1-4GlcNAc) it was possible to compare profiles quantitat ively. Although no peak was identified as disease-specific, characteri stic and reproducible profiles were obtained. Particularly striking we re reductions in the major peaks in Crohn's disease, rheumatoid arthri tis, stomach cancer, accompanied by increases in unidentified peaks. P revious studies suggested that many of the unknown peaks were due to i ncreased 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.