DETERMINATION OF SINGLE MONOSUGARS BOUND TO A PEPTIDE

A method is described which allows detection and quantitative determin ation of single monosugar units bound O-glycosidically to a peptide. A glycoprotein or a glycopeptide is chemically degraded under the modif ied conditions of Carlson degradation (beta-elimination performed in w eakly alkaline conditions in the presence of sodium borohydride). An a liquot of the neutralized reaction mixture, supplemented with an inter nal 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 degree s C only monosugar peracetylated alditols reach the detector. By compa ring the retention times of appropriate peaks with standards and by ch ecking their mass spectra the monosugar alditols are unequivocally ide ntified. The detectable amount of a reduced monosugar in the analyzed sample is about 0.3 mu g. Several glycoproteins were analyzed using th is method. Free N-acetylgalactosaminitol was detected in the degradati on products of human glycophorin A and ovine submaxillary mucin, addit ionally free galactitol was detected in the degradation products of gl ycophorin. This result suggests that some single galactose units, O-gl ycosidically linked to the peptide are present in human glycophorin A.