Permethylation for glycan analysis

Authors：

Category

Isolation & structural analysis of glycans

Protocol Name

Authors

Kameyama, Akihiko
Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST)

KeyWords

solid phase extraction permethylation mass spectrometry

Reagents

●	Iodomethane, Wako Special Grade, (Wako Pure Chemical Industries Ltd., Osaka, Japan)
●	Dimethyl sulfoxide, Infinity Pure Grade, (Wako Pure Chemical Industries Ltd.)
●	Sodium hydroxide, purum p.a., ≥98.0% (T), beads (small), (Fluka, Deisenhofen, Germany)

Instruments

●	Agate mortar
●	Small glass tube with a screw cap, conical bottom
●	Vortex mixer
●	Solid phase extraction device (Sep-Pak C-18; Waters Corp., Milford, MA)

Methods

Permethylation for glycan analysis

* This procedure describes the small scale permethylation of glycans (<50 μg).

1)	An aliquot of glycan solution was transferred to a small glass tube*, and then evaporated to dryness using a centrifugal evaporator.	Comment 1

2)	A small amounts of sodium hydroxide and DMSO containing 1% (vol/vol) distilled water* were placed in an agate mortar, and grinded to a slurry. (Fig. 1)	Comment 1

3)	Ca. 50 μL of the slurry was transferred to the glass tube containing the glycans*. (Fig. 2 a and b)	Comment 1

4)	Ca. 50 μL of methyl iodide* was added to the glass tube. (Fig. 2 c)	Comment 1

5)	The tube was securely capped and the mixture vigorously shaken at high speed using a vortex mixer at room temperature for 15 min*.	Comment 1

6)	1% HCl (1 mL) was slowly added to the reaction mixture, and then the mixture was applied to an Sep-Pak C-18 (50 mg, syringe type:1 cc) cartridge*.	Comment 1

7)	The Sep-Pak Cartridge was washed with distilled water (3 mL).	Comment 0

After the washing, the permethylated glycans were eluted with 80% (vol/vol) acetonitrile (1 mL), and evaporated to dryness by centrifugal evaporator.

Comment 0

Notes

Safety concerns

This procedure should be performed in a fume hood.

Wear suitable protective eyewear and gloves. Do not use latex gloves, which are permeable to methyl iodide. Nitrile or poly chloroprene gloves are recommended.

Acknowledgement

Author thanks Drs K-H. Khoo and S-Y. Yu for training on this practical handling for permethylation.

Figure & Legends

Fig. 1. Preparation of NaOH slurry

(a) small beads of NaOH in an agate mortar, (b) addition of DMSO containing 1% (vol/vol) distilled water, (c) grinding the NaOH beads to a slurry.

Fig. 2. Transfer the reagents into the reaction tube

(a) measuring off the NaOH slurry using a micropipette, (b) the reaction tube containing the slurry, (c) measuring off methyl iodide using a micro syringe.

Fig. 3. Cutting the end off a pipette tip

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Date of registration：2014-06-19 13:46:36