Method of isolating and analyzing oligosaccharides in glycoproteins

摘要

A method for isolating derivatives of general O-linked amino acids including derivatives of O-tyrosine linked kappa casein di-O-sulfo tetrasaccharide and of O-di-phospho serine linked bovine submaxillary mucin disaccharide is provided. These derivatives are isolated and analyzed by further enzymatically separating the N-linked oligosaccharides from the O-linked oligosaccharides and then cleave O-linked oligosaccharides by decreasing the pH levels such that de-amidations are allowed. This de-amidation results in individual O-linked glycan amino acid components removed from the original glycoprotein. The O-linked glycan amino acid components are thus isolated from the glycoprotein, and can be individually analyzed.

主权项

1. A method of oligosaccharide isolation and analysis comprising the steps of:
treating a glycoprotein sample to cleave N-linked oligosaccharides from the sample to form a first solution containing a residual protein; passing the first solution in an ammonium form cation exchange resin within an ion exchange cartridge by washing the cartridge with a predetermined amount of ammonium hydroxide solution to form a second solution eluted from the cation exchange resin; adding a predetermined amount of sodium borohydride to the second solution to form a third solution; letting the third solution stand capped for a predetermined amount of time; letting the third solution stand uncapped for a predetermined amount of time; evaporating the third solution to a predetermined volume to form a fourth solution; mixing the fourth solution with water and passing the fourth solution through a sodium form cation exchange resin within an ion exchange cartridge and collecting an eluent off the ion exchange cartridge; and analyzing the eluent with an analysis device.