Determination of single monosugars bound to a peptide.

A method is described which allows detection and quantitative determination of single monosugar units bound O-glycosidically to a peptide. A glycoprotein or a glycopeptide is chemically degraded under the modified conditions of Carlson degradation (beta-elimination performed in weakly alkaline conditions in the presence of sodium borohydride). An aliquot of the neutralized reaction mixture, supplemented with an internal standard, is peracetylated, extracted and directly analyzed by g.l.c.-m.s. All the O-linked oligosaccharides split off from the peptide are derivatized, but under gas-liquid chromatography at 150-230 degrees C only monosugar peracetylated alditols reach the detector. By comparing the retention times of appropriate peaks with standards and by checking their mass spectra the monosugar alditols are unequivocally identified. The detectable amount of a reduced monosugar in the analyzed sample is about 0.3 microgram. Several glycoproteins were analyzed using this method. Free N-acetylgalactosaminitol was detected in the degradation products of human glycophorin A and ovine submaxillary mucin, additionally free galactitol was detected in the degradation products of glycophorin. This result suggests that some single galactose units, O-glycosidically linked to the peptide are present in human glycophorin A.

Monoclonal Antibodies Recognising Sialyl-Tn: Production and Application to Immunochemistry

In order to develop reagents that can detect the exposed sialyl-Tn antigen (NeuAcα2,6GaINAcα 1-O-Ser/Thr) on tumour-associated mucins, we have prepared monoclonal antibodies (mabs 3C2 and 301, both IgM) against ovine submaxillary mucin (OSM; >98% of glycans as sialyl-Tn). These mabs showed strong reactivity with OSM and bovine submaxillary mucin (BSM; 50% of glycans as sialyl-Tn) but did not react with desialylated OSM or BSM. Sialic acid at I mg/ml did not significantly inhibit mab binding to OSM, suggesting that the linkage to GalNAc may be important for mab binding. 3C2 and 3D I also showed similar reactivity to sialyl-Tn reactive mab Bn.3, and detected Bn.3 capturedOSM in a sandwich ELISA. In Western blotting of mucus from a patient with a mucinous ovarian tumour, the mabs reacted with high molecular weight (>200 kDa) species. In immunohistochemistry, these mabs showed strong reactivity with most cancers of the colon, lung, and stomach, and also some tumours of the ovary and breast. There was only limited reactivity in normal tissue from these sites. The antibodies should be useful reagents for the detection of the sialyl-Tn antigen in human cancers.