ovalbumin and hydrazine

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.

Topics: alpha-Fetoproteins; Amidohydrolases; Carbohydrate Sequence; Chromatography, High Pressure Liquid; Glycoproteins; Hydrazines; Magnetic Resonance Spectroscopy; Molecular Sequence Data; N-Acetylneuraminic Acid; Neuraminidase; Oligosaccharides; Ovalbumin; Oxidation-Reduction; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Pyrazoles; Pyrazolones; Ribonucleases; Sialic Acids; Thyroglobulin; Trypsin