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

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.

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

PAGE and ESI-FTMS analysis of CE-PAGE purified heparin oligosaccharides (insets): A. tetrasaccharide, dp4; and B. tetradecasaccharide, dp14.

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

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.