A straightforward protocol, which employs techniques like microwave-assisted enzymatic or chemical cleavage of glycoproteins, high performance liquid chromatography (HPLC), mass spectrometry, and microwave-assisted partial acid hydrolysis of glycopeptide, to elucidate oligosaccharide composition of glycoprotein is presented here. Glycoprotein ribonuclease B (RNase B) is used as a model system to illustrate the features of this protocol. Cyanogen bromide (CNBr) or trypsin cleavage provides an effective way of cutting RNase B into small manageable glycopeptides and this reaction can be sped up by a short irradiation to microwaves. The reducing agent tris(2-carboxyethyl)phosphine hydrochloride was effective in unfolding the protein, which facilitates the CNBr or trypsin cleavage. Glycopeptides were easily purified using reversed-phase C18 HPLC. In addition, partial hydrolysis of oligosaccharide moiety of glycopeptides using trifluoroacetic acid or 6 M HCl was observed after one minute of microwave irradiation. The compositions of oligosaccharides in RNase B were readily derived using high-resolution mass spectrometry and the data agrees with the proposed oligosaccharide structures of RNase B (five glycoforms). This method makes the analysis of glycoproteins with molecular weights greater than 30 kDa much simpler and achievable.
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