Measurement of amino acid compositions of glycoprotein systems by gas-phase hydrolysis and reversed-phase high-performance liquid chromatography

Interest in glycoproteins and their compositions has increased in recent years. Work described in this report illustrates the use of an amino acid analysis protocol involving gas-phase hydrolysis and reversed-phase high-performance liquid chromatography of glycoprotein systems at microgram levels. In other amino acid analysis protocols the problem of losses of amino acids of glycoproteins has been documented. These losses were due to various reactions, referred to as browning or Maillard reactions, which yielded a residue from which amino acids were not recoverable. In our work, three glycoprotein systems are examined: ovalbumin, sICAM-1, and bovine serum albumin--which is naturally unglycosylated, but is spiked with about 30% saccharides. In all three cases, the compositional agreement between the molar ratio of amino acids determined empirically and that predicted is greater than 90%. Thus it is shown that the adverse effects of Maillard-type reactions are avoided, and the presence of carbohydrates causes negligible interferences with amino acid analysis performed under the conditions described herein.