Intact and Oligomeric Glycosaminoglycans Analyzed with Capillary Electrophoresis

Nikos K. Karamanosand Anders Hjerpe

1. Introduction

Various types of capillary electrophoresis (CE) can be used to study native and oligomeric glycan structures. For a recent review, see ref. 1. These separations include not only capillary zone electrophoresis (CZE), discussed in Chapter 18, but also capillary gel electrophoresis (CGE). This can be performed with the capillary filled with a cross-linked chemical gel as in common polyacrylamide electrophoresis. Alternatively, a neutral polymer (pullulan, PEG, dextran, etc.) can be added to the operating buffer, creating a matrix that retards the analyte in a similar way, depending on its molecular size. Although it is not described in this chapter, micellar electrokinetic capillary chromatography (MECC) may also be valuable for the study of glycosaminoglycan (GAG) fragments (2). In MECC, the separation buffer contains surfactant in a concentration sufficient for the formation of micelles that migrate more slowly than the electroos-motic (EOF) or in the opposite direction. This creates a situation similar to that in reversed-phase chromatography, with the micelles as the stationary phase. The separation of analytes depends on differences in the partition between micelles and buffer.

Several biologically important interactions of proteoglycans (PGs) require GAG structures longer than a disaccharide, the size of this sequence in several cases being around 5 monosaccharides or more. A limitation in establishing such functional oligomeric sequences and their fine structures has been a lack of techniques that sufficiently separates various larger GAG fragments. The high-resolution efficiency of the CE-based techniques permits such separations (3), and this technique can, after further methodological development, become an important tool in such a context.

In the present chapter, we outline protocols for the separation of different GAGs, depending on charge density (hyaluronan [HA], chondroitin sulfate [CS], dermatan sulfate [DS], heparan sulfate [HS], and heparin) (4) and visualization of molecular

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

size and polydispersity (HA) (5,6). CE can also be used to demonstrate and identify specific oligosaccharide sequences (3). This is of value for the structural characterization of GAGs, and a procedure for such separations is provided in the Chapter 18, Subheading 3.2.

2. Material

2.1. CZE of Different GAGs Related to Charge Density

1. Standard GAGs: Prepare a stock solution (1.0 mg/mL) of HA, CSA (CS sulfated mainly at C-4), or CSC (CS sulfated mainly at C-6), DS, HS, heparin and/or Fragmin® (a fragment of heparin with a molecular size of 5000 daltons) by dissolving the substances in the operating buffer. Make serial dilutions (1/1,000, 1/300, and 1/100) in the operating buffer so as to prepare standard GAG solutions of 1.0, 3.3, and 10.0 ^g/mL.

2. Capillary: Uncoated fused-silica (75 ^m id, 50 cm effective length).

3. Operating buffer: 20 mM orthophosphate buffer, pH 3.0. The buffer is passed through a 0.2-^m membrane filter, divided in portions of 1 mL, and kept at -20°C.

4. 0.1 M NaOH, prepared in 2 x distilled water and passed through a 0.2-^m membrane filter.

2.2. CGE of HA Using Pullulan Matrix

1. Operating buffer: 50 mM phosphate buffer, pH 4.0, is passed through a 0.2-^m membrane filter, whereafter 0.10% (w/v) pullulan with a molecular weight of 1,600,000 is added (see Note 1).

2. Capillary: the separation is performed in a 75-^m-id uncoated fused-silica capillary with an effective length of 50 cm.

2.3. CGE of HA Oligomers Using PEG Matrix

1. Operating buffer: 0.1 M Tris-HCI/0.25 M borate, pH 8.5, containing 10% (w/v) polyethylene glycol (PEG) with average molecular weight of 70,000 (PEG70000).

2. The separation is performed in a 100-^m-id fused-silica capillary coated with (50% phenyl)methyl-polysiloxane, effective length 20 cm.

3. Methods

3.1. CZE of Different GAGs Related to Charge Density

GAG preparations can be analyzed for the homogeneity of their charge density by simple CZE using detection at low wavelength. Following the protocol described below, HA, CS/DS, HS, and HA are separated at low pH using reversed polarity. CSA, CSB, and DS migrate almost identically since they have similar charge densities. Heparin and Fragmin migrate first due to their high content of sulfates, whereas HA migrates last since it is a nonsulfated GAG (see Fig. 1).

1. Dissolve the samples containing approximately 0.1-10 ^g of GAGs in 100 ^L of operating buffer.

2. Start the CE instrument and adjust the detector wavelength at 190 nm (see Note 2).

3. Prepare the software of the CE instrument as follows.

a. Rinse the capillary with 0.1 M NaOH for 1 min.

b. Condition the capillary with operating buffer for 4 min.

c. Inject the sample at the cathode (reversed polarity), using the pressure mode (500-700 mbar x s).

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