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

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• 作者：Benjamin L. Schulz ; Nicolle H. Packer ; and Niclas G. Karlsson
• 刊名：Analytical Chemistry
• 出版年：2002
• 出版时间：December 1, 2002
• 年：2002
• 卷：74
• 期：23
• 页码：6088 - 6097
• 全文大小：246K
• 年卷期：v.74,no.23(December 1, 2002)
• ISSN：1520-6882

文摘

A technique with subpicomolar sensitivity was developedfor analyzing O-linked oligosaccharides released fromglycoproteins separated by gel electrophoresis. The protocol involves gel electrophoresis, electroblotting to poly(vinylidene fluoride) membrane, reductive ges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-elimination,and analysis of released oligosaccharides by liquid chromatography coupled to negative ion electrospray massspectrometry. 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 proteinbackbone, giving some information on site-specific glycosylation. The method was used to demonstrate that thedifference between human ges/gifchars/alpha.gif" BORDER=0>-2HS-glycoprotein isoformsseparated by 2D-gel electrophoresis was partially due tosialylation of both O-linked and N-linked oligosaccharides.It was also shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneouslyfrom high molecular mass (200 000-5 000 000 Da)highly glycosylated mucin glycoproteins collected fromsmall intestine and saliva and separated by sodiumdodecyl sulfate-agarose/polyacrylamide composite gels.Mass spectrometric data not only gave information aboutthe mass distribution of the heterogeneous mixtures ofoligosaccharides from [M - xH]^x- ions but also gaveinformation about the isomeric heterogeneity of the oligosaccharides from their resolution by porous graphitizedcarbon chromatography. Tandem mass spectrometry wasexplored as a technique for distinguishing between oligosaccharide isomers with different sequences and alsobetween oligosaccharides with the same sequence butwith different linkage configurations.