Chromatographic separation of monosaccharides hydrolyzed from glycoconjugates or complex, aggregate biomaterials, can be achieved by classic analytical methods without a need for derivatizing the monosaccharide subunits. A simple and sensitive method is presented for characterizing underivatized monosaccharides following hydrolysis from N- and O-linked glycoproteins using high-performance liquid chromatography separation with mass spectrometry detection (LC-MS). This method is adaptable for characterizing anything from purified glycoproteins to mixtures of glycoforms, for relative or absolute quantification applications, and even for the analysis of complex biomaterials. Use of an amide stationary phase and HILIC chromatography is demonstrated to retain the highly polar, underivatized monosaccharides and resolve stereoisomers and potentially interfering contaminants. This work illustrates an original approach for qualitative characterization of N- and O-linked glycoprotein standards, mixtures, and for complex biological materials such as a total yeast extract.
monosaccharides, liquid chromatography, multiple-reaction monitoring (MRM), hydrolysis, glycosylation