Small-Scale Analysis of O-Linked Oligosaccharides from Glycoproteins and Mucins Separated by Gel Electrophoresis

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Abstract

A technique with subpicomolar sensitivity was developed for analyzing O-linked oligosaccharides released from glycoproteins separated by gel electrophoresis. The protocol involves gel electrophoresis, electroblotting to poly-(vinylidene fluoride) membrane, reductive beta-elimination, and analysis of released oligosaccharides by liquid chromatography coupled to negative ion electrospray mass spectrometry. It was also found that N-linked oligosaccharides could be recovered under the same conditions, found both as free oligosaccharides and as distinct glycopeptides created from reductive cleavage of the protein backbone, giving some information on site-specific glycosylation. The method was used to demonstrate that the difference between human alpha-2HS-glycoprotein isoforms separated by 2D-gel electrophoresis was partially due to sialylation of both O-linked and N-linked oligosaccharides. It was also shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneously from high molecular mass (200,000-5,000,000 Da) highly glycosylated mucin glycoproteins collected from small intestine and saliva and separated by sodium dodecyl sulfate-agarose/polyacrylamide composite gels. Mass spectrometric data not only gave information about the mass distribution of the heterogeneous mixtures of oligosaccharides from [M - xH](x-) ions but also gave information about the isomeric heterogeneity of the oligosaccharides from their resolution by porous graphitized carbon chromatography. Tandem mass spectrometry was explored as a technique for distinguishing between oligosaccharide isomers with different sequences and also between oligosaccharides with the same sequence but with different linkage configurations.

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