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. 2010 Jun 15;401(2):236-41.
doi: 10.1016/j.ab.2010.03.004. Epub 2010 Mar 6.

High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis

Tatiana N Laremore  1 , Mellisa Ly, Kemal Solakyildirim, Dmitri V Zagorevski, Robert J Linhardt
Affiliations

High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis

Tatiana N Laremore et al. Anal Biochem. .

Abstract

Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6h using continuous elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies.

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Conflict of interest statement

Conflict of interest

None declared

Figures

Figure 1
Figure 1
Structure of bovine lung heparin. An average chain contains major trisulfated disaccharide repeating units (n ~ 27) and minor disaccharide repeating units (m ~ 3) where X = H or SO3− and Y = SO3− and occasionally COCH3 or H.
Figure 2
Figure 2
PAGE and ESI-FTMS analysis of CE-PAGE purified heparin oligosaccharides (insets): A. tetrasaccharide, dp4; and B. tetradecasaccharide, dp14.
Figure 3
Figure 3
1H-NMR analysis of A. BLH oligosaccharide mixture; B. gel-eluted dp6 + dp8 fraction after desalting using 3,000 MWCO spin-column; and C. gel-eluted dp6 + dp8 fraction after purification using SAX spin-column.
Figure 4
Figure 4
Two-dimensional NMR analysis of BLH oligosaccharide mixture (A, B) and CE-PAGE purified BLH decasaccharide, dp10 (C, D). The NMR peaks corresponding to H-H (COSY, panels A,C) or H-C (HMQC, panels B, D) correlations are labeled N for GlcN and I for IdoA residues, and A for non-reducing end unsaturated uronic acid residue.
See this image and copyright information in PMC

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