Nikos K. Karamanosand Anders Hjerpe
During the past decade, the use of fully automated equipment for capillary electrophoresis (CE) has made this a routine method for the study of soluble analytes. The separation of such compounds in capillaries (20-200 ^m id) and in strong electric fields (around 50 kV/m) seems to be exceptionally efficient for separating both large and small molecules. The most common type of CE is capillary zone electrophoresis (CZE), which is done with the separation buffer free in an otherwise empty capillary. These separations, in principle, share some features not only with gel electrophoresis but also with high-performance liquid chromatography (HPLC), and they provide a unique combination of both analytical techniques. CZE is therefore an alternative to HPLC and it is sometimes advantageous because there are no problems related to laminar flow and other wall effects.
Other advantages of CZE, compared to HPLC and gel electrophoresis, are that it is more friendly to the user and the environment. Only minute amounts of solvents are needed, and the use of aqueous separation buffers in open capillaries eliminates the need for toxic organic solvents and acrylamide.
The migration of analytes toward the detector depends on two main factors: electro-phoretic mobility (EM), due to the net charge of the analyte; and electroosomotic flow (EOF) of the free solution, caused mainly by dissociation of silanol groups in the capillary glass wall and migration of resultant H3O+ toward the anode. Both these effects can be modulated to change the separation. The net charge of the analyte is readily modified by ion pairing or ion suppression. The EOF of the solution depends on the pH and can be completely blocked by the inclusion of detergents in the separation buffer or by coating the inner capillary surface with hydrophobic agents or surfactants.
From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ
Modern detectors yield an electropherogram that is similar to a chromatogram. The ideal flow characteristics and resultant high resolution provide a considerably higher sensitivity than HPLC in terms of analyte amounts, while the short light paths through the detection window of the capillary often necessitate concentrations similar to those of HPLC. Stacking of injected material, however, sometimes creates deviations from this general rule to give exceptional sensitivity (1). Another way to improve the sensitivity is to derivatize the analytes with fluorescent tags, allowing the use of laser-induced fluorescence (LIF) detection (2,3).
The proteoglycans (PGs) with their glycosaminoglycan (GAG) side chains are involved in a wide range of biological processes. Although the GAGs are synthesized to yield only a few main types, the possible variations in structure are enormous (4). Our knowledge of the structural background of specific GAG interactions and their biological importance increases with improved methods for elucidation of the fine structure. In such studies, CE can be powerful as an alternative tool or complement to other analytical techniques. The various methods developed in this context have recently been reviewed (5,6).
In this chapter we present protocols for the analysis of A-disaccharides obtained from digestion of GAGs with specific lyases. These separations can be used to obtain information on the total amounts of hyaluronan (HA) and galactosaminoglycans (GalAGs)—i.e., chondroitin sulfate (CS) and dermatan sulfate (DS) (7)—and the disac-charide composition of these GAGs, the latter including the type of uronic acid in DS, sulfation patterns in CS/DS (1) or heparan sulfate (HS)/heparin, and the extent of N-acetyla-tion in heparin/HS (8,9). A strategy for analyzing all these GAGs is shown in Fig. 1.
To our knowledge, there are no CZE-based methods for analyzing keratan sulfate disaccharide composition, although the availability of specific keratanases would make such separations possible.
2.1. Total Content of HA and GalAGs
1. Digestion buffer: 25 mM Tris-HCl, pH 7.5. The buffer is passed through a 0.2-^m membrane filter and kept at -20°C pending use.
2. Standard HA/CS solution: Prepare stock solutions with HA and CS (preferably use CSA of mammal origin), each containing 1.0 mg/mL. Determine the exact GAG content in each solution colorimetrically (for example, by the carbazole reaction). Make a standard solution with 1 mL of each stock solution and 8 mL of the digestion buffer. Divide in portions and store at -20°C until use.
3. HA/GalAGs degradation buffer: Dissolve chondroitinases ABC and AC as well as chondrosulfatases-4 and -6 in the digestion buffer, so as to give 1 unit/mL. An equi-unit mixture of all lyases is then prepared by mixing 10 ^L of each enzyme to produce 40 ^L of the buffer containing 0.01 unit of each enzyme (see Note 1).
4. Capillary: Uncoated fused-silica (75-^m id, effective length 50 cm) (see Note 2).
5. Operating buffer: 15 mM sodium orthophosphate buffer, pH 3.0. The buffer is passed through a 0.2-^m membrane filter, divided into portions of 1 mL, and kept at -20°C pending use (see Note 3).
6. 0.1 M NaOH, prepared in 2 x distilled water and passed through a 0.2-^m membrane filter.
7. Centricon 3 membrane (cutoff 3000 daltons) microfuge tubes.
2.2. Structural Characterization of GalAGs
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