Fluorescent labelling of glycans and HPLC analysis

Authors：

Category

Isolation & structural analysis of glycans

Protocol Name

Authors

Nakakita, Shin-ichi *
Department of Functional Glycomics, Life Science Research Center, Kagawa University

Natsuka, Shunji
Department of Biology, Faculty of Science, Niigata University

*To whom correspondence should be addressed.

KeyWords

Fluorescent label Pyridylamination Reducing amination

Reagents

●	2-aminopyridine (Nacalai Tesque, Inc., Kyoto, Japan)
●	dimethylamine-borane (Wako Pure Chemical Industries Ltd., Osaka, Japan)
●	acetic acid (Wako Pure Chemical Industries Ltd.)
●	25% ammonia solution (Wako Pure Chemical Industries Ltd.)
●	1-butanol (Wako Pure Chemical Industries Ltd.)
●	acetonitrile (Wako Pure Chemical Industries Ltd.)
●	chloroform (Wako Pure Chemical Industries Ltd.)
●	phenol (Wako Pure Chemical Industries Ltd.)

Instruments

●	C18 Sep-Pak cartridge (Waters Corp., Milford, MA)
●	HPLC connected to fluorescence detector (Waters Corp.)
●	Speed Vac Concentrator (Thermo Fisher Scientific Inc., Waltham, MA)
●	Block incubator (Astec Co., Ltd., Fukuoka, Japan)
●	Mono-Q 5 × 50 mm (GE Healthcare, Little Chalfont, UK)
●	Cosmosil 5C18P 4.6 × 150 mm (Nacalai Tesque, Inc.)
●	Shodex Asahipak NH2P-50 4.6 × 50 mm (Showa Denko K.K., Tokyo, Japan)

Methods

Fluorescent labelling of glycan and HPLC analysis

1)	Infuse saccharide solution in conical micro tube.	Comment 0

2)	Lyophilize sample.	Comment 0

3)	Add 0.02 mL of the coupling reagent solution (552 mg of 2-aminopyridine dissolved in 0.2 mL acetic acid).	Comment 0

4)	Mix coupling reagent solution and sample.	Comment 0

5)	Heat at 90°C, 1 h.	Comment 0

6)	Add 0.07 mL of the reducing reagent solution (200 mg of borane-dimethylamine complex dissolved in 0.08 mL of acetic acid and 0.05 mL of water).	Comment 0

7)	Mix reducing reagent solution and sample.	Comment 0

8)	Heat at 80°C, 35 min.	Comment 0

9)	Mix 0.09 mL water and 0.09 mL phenol/chloroform solution.	Comment 0

10)	Centrifuge 200 × g, 1 min.	Comment 0

11)	Remove organic phase.	Comment 0

12)	Repeat 9–11 step twice.	Comment 0

13)	Lyophilize water phase.	Comment 0

14)

Analyze by HPLCs(Reversed-phase, size-fractionation, anion-exchange).

Comment 1

Figure & Legends

Fig. 1. Scheme of pyridylamination

This figure was originally published in "Mirai wo Hiraku Tousa Kagaku" edited by Nagai K, Kawasaki T. Kinpodo. 2005, pp.10–11 (Section 1.6, Nakakita S.).

Fig. 2. Gradient programs of HPLC using of PA-oligosaccharide analysis

1. Anion exchange HPLC (MonoQ), A buffer: ammonia solution (pH 9.0); B buffer: 500 mM ammonium-acetate buffer (pH 9.0), flow rate 1 mL/min, 25˚C.

2. Size-fractionation HPLC (Shodex Asahipak NH2P-50), 50 mM ammonium-acetate buffer (pH 7.0) in 93% acetonitrile; B buffer: 50 mM ammonium-acetate buffer (pH 7.0) in 20% acetonitrile, , flow rate 0.6 mL/min, 25˚C.

3. Reversed-phase HPLC of N-glycan mode (Cosmosil 5C18P,), 100 mM ammonium-acetate buffer (pH 4.0); B buffer: 100 mM ammonium-acetate buffer (pH 4.0) in 0.5% 1-butanol, flow rate 1.5 mL/min, 25˚C.

4. Reversed-phase HPLC of O-glycan mode (Cosmosil 5C18P), 100 mM ammonium-acetate buffer (pH 6.0); B buffer: 100 mM ammonium-acetate buffer (pH 6.0) in 1% 1-butanol, flow rate 1.5 mL/min, 25˚C.

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Date of registration：2015-05-08 15:03:27