HPLC analysis of glycosaminoglycans

Authors：

Category

Glycosaminoglycans

Protocol Name

Authors

Yamada, Shuhei *
Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University

Toyoda, Hidenao
Laboratory of Bio-analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University

*To whom correspondence should be addressed.

KeyWords

chondroitinase heparinase fluorescence

Reagents

●	Chondroitinase ABC (a conventional preparation) from Proteus vulgaris (Seikagaku Corp., Tokyo, Japan)
●	Chondroitinase AC-II from Arthrobacter aurescens (Seikagaku Corp.)
●	Heparinase from Flavobacterium heparinum (Seikagaku Corp.)
●	Heparitinases I and II from Flavobacterium heparinum (Seikagaku Corp.)
●	Unsaturated chondro-disaccharide kit (C kit) (for HPLC) (Seikagaku Corp.)
●	Unsaturated HS/HEP-disaccharide kit (H kit) (for HPLC) (Seikagaku Corp.)
●	2-Aminobenzamide (2AB) (Nacalai Tesque Inc., Kyoto, Japan)
●	NaCNBH₄ (Sigma-Aldrich, St. Louis, MO)
●	2-Cyanoacetamide (Sigma-Aldrich)
●	Tetra-n-butylammonium hydrogen sulfate (Sigma-Aldrich)

Instruments

●	YMC-Pack PA-03 (4.6 mm i.d. x 250mm) (YMC Co., Ltd., Kyoto, Japan)
●	Senshu Pak DOCOSIL A column (4.6 mm i.d. x 150 mm, 5 μm) (Senshu Scientific Co., Ltd., Tokyo, Japan)
●	Speed Vac Concentrator (Thermo Fisher Scientific Inc., Waltham, MA)
●	Ultrafree-MC (Merck Millipore, Billerica, MA)
●	HPLC system (LC-20 Series: Shimadzu Corp., Kyoto, Japan)
●	HPLC system (L-2000 Series: Hitachi High-Tech Science Corp., Tokyo, Japan)

Methods

HPLC analysis of GAG di- and oligosaccharides (precolumn-labeling)

1)	Enzymatic digestion of GAGs: Incubate heparin (Hep)/heparan sulfate (HS) (25-250 ng) with a mixture of heparinase and heparitinase (1 mIU each) in a total volume of 20 μL of 20 mM acetate-Na buffer, pH 7.0, containing 2 mM Ca(CH₃COO)₂ at 37˚C for 1 h. Incubate chondroitin sulfate (CS)/dermatan sulfate (DS) or hyaluronan (HA) (25-250 ng) with a mixture of chondroitinases ABC and AC-II (1 mIU each) in a total volume of 20 μL of 50 mM acetate-Na buffer, pH 6.0, at 37˚C for 30 min.	Comment 1

2)	Terminate the reactions by boiling at 100 ˚C for 1 min.	Comment 0

3)	Lyophilize the sample by Speed Vac.	Comment 0

4)	Add 5 μL of the 2AB-derivatization reagent mixture (0.35 M 2AB/1.0 M NaCNBH₄/30% (v/v) acetic acid in dimethyl sulfoxide) and incubate at 65˚C for 2 h to label the reducing end with a fluorophore.	Comment 1

5)	Dilute the reaction mixture with distilled water to 200 μL and transfer it to a glass tube.	Comment 0

6)	Remove the excess 2AB reagent by extraction with an equivalent volume of chloroform 5 times.	Comment 0

7)	Analyze an aliquot (40 μL) of the aqueous phase by HPLC on an amine-bound silica PA-03 column using a linear gradient of NaH₂PO₄ from 16 to 530 mM at a flow rate of 1 mL/min, monitored by measuring fluorescence with excitation and emission wavelengths of 330 and 420 nm, respectively.	Comment 0

Identify and quantify the eluted peaks by comparing with the elution positions of authentic standards (Fig. 1 and 2).

Comment 0

HPLC analysis of GAG di- and oligosaccharides (postcolumn-labeling)

1)	Enzymatic digestion of GAGs: Incubate Hep/HS (1-250 ng) with a mixture of heparinase and heparitinases I and II (1 mIU each) in a total volume of 15 μL of 25 mM sodium acetate buffer, pH 7.0, containing 2 mM calcium acetate at 37˚C for 16 h. Incubate CS/DS or HA (1-250 ng) with a mixture of chondroitinases ABC and AC-II (5 mIU each) in a total volume of 15 μL of 25 mM Tris-acetate buffer, pH 8.0, at 37˚C for 3 h.	Comment 0

2)	Terminate the reactions by boiling at 100˚C for 1 min.	Comment 0

3)	Dry the sample by Speed Vac.	Comment 0

Add 10 μL of distilled water, then inject 8 μL of the sample into the postcolumn HPLC system. The flow diagram and HPLC conditions are shown in Fig. 3. Typical chromatograms are shown in Fig. 4.

Comment 0

Figure & Legends

Fig. 1. Structure of unsaturated disaccharides

The structure of unsaturated disaccharides derived from HS/Hep (A) as well as CS/DS and HA (B) is given. Treatment with bacterial eliminases converts the original uronic acid structure in the polysaccharides into an artificial structure, the 4,5-unsaturated uronic acid, 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid (ΔHexA). GalNAc, GlcNAc, GlcN, 2S, 4S, 6S, and NS represent N-acetylgalactosamine, N-acetylglucosamine, glucosamine, 2-O-sulfate, 4-O-sulfate, 6-O-sulfate, and 2-N-sulfate, respectively.

Fig. 2. HPLC profile of the 2AB-labeled unsaturated disaccharide standards

Elution positions of the 2AB-derivatives of the standard disaccharides derived from HS/Hep (A) as well as CS/DS and HA (B) are shown. 1, ΔHexAα1-4GlcNAc; 2, ΔHexAα1-4GlcNAc(6S); 3, ΔHexAα1-4GlcN(NS); 4, ΔHexAα1-4GlcN(NS, 6S); 5, ΔHexA(2S)α1-4GlcN(NS); 6, ΔHexA(2S)α1-4GlcN(NS, 6S); a, ΔHexAα1-3GlcNAc; b, ΔHexAα1-3GalNAc; c, ΔHexAα1-3GalNAc(6S); d, ΔHexAα1-3GalNAc(4S); e, ΔHexA(2S)α1-3GalNAc(6S); f, ΔHexA(2S)α1-3GalNAc(4S); g, ΔHexAα1-3GalNAc(4S, 6S); h, ΔHexA(2S)α1-3GalNAc(4S, 6S). The HPLC analysis was carried out on an amine-bound silica PA-03 column under isocratic conditions with 16 mM of NaH₂PO₄ for the first 10 min followed by a linear gradient from 16 to 538 mM NaH₂PO₄over 60 min.

Fig. 3. Flow diagram of the postcolumn HPLC system for analyzing the unsaturated disaccharides

The sample is separated on a C22 reversed phase column (4.6 mm i.d. x 150 mm) at 55˚C with a NaCl gradient (flow rate, 1.1 mL/min). The column eluate is then mixed with 0.5% 2-cyanoacetamide and 1.0% NaOH, supplied by the double-plunger pump (flow rate, 0.35 mL/min), and reacted at 125˚C to form fluorescence products. Reaction coil, 0.5 mm i.d. x 10 m; cooling coil, 0.25 mm i.d. x 2 m; detection, excitation 346 nm, emission 410 nm. Solvent A is 1.2 mM tetra-n-butylammonium hydrogen sulfate in 8.5% acetonitrile. Solvent B is 0.2 M NaCl in solvent A. Gradient program: 0-10 min, 1-4% B; 10-11 min 4-15% B; 11-20 min, 15-25% B; 20-22 min, 25-53%; 22-29 min, 53% B; equilibration with 1% B for 20 min.

Fig. 4. Typical chromatograms of unsaturated disaccharides derived from CS/DS (A) and HS/Hep (B)

(A) Sample size (5 μL, 5 ng of each sugar). Peaks: 1, ΔHexAα1-3GalNAc; 2, ΔHexAα1-3GalNAc(4S); 3, ΔHexAα1-3GalNAc(6S); 4, ΔHexA(2S)α1-3GalNAc; 5, ΔHexAα1-3GalNAc(4S, 6S); 6, ΔHexA(2S)α1-3GalNAc(4S); 7, ΔHexA(2S)α1-3GalNAc(6S); 8, ΔHexA(2S)α1-3GalNAc(4S, 6S). (B) Sample size (5 μL, 5 ng of each sugar). Peaks: 9, ΔHexAα1-4GlcNAc; 10, ΔHexAα1-4GlcN(NS); 11, ΔHexAα1-4GlcNAc(6S); 12, ΔHexA(2S)α1-4GlcNAc; 13, ΔHexAα1-4GlcN(NS, 6S); 14, ΔHexA(2S)α1-4GlcN(NS); 15, ΔHexA(2S)α1-4GlcNAc(6S); 16, ΔHexA(2S)α1-4GlcN(NS, 6S).

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Date of registration：2014-03-31 11:34:22