N-Glycan profiling by LC/MS

Authors：

Category

Isolation & structural analysis of glycans

Protocol Name

Authors

Kuribayashi, Ryosuke
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences

Nakazawa, Shiori
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences

Kawasaki, Nana *
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences

*To whom correspondence should be addressed.

KeyWords

N-glycans glycan profile LC/MS

Reagents

●	N-Glycosidase F (Roche Diagnostics GmbH, Mannheim, Germany)
●	Sodium tetrahydroborate (NaBH_4,Wako Pure Chemical Industries Ltd., Osaka, Japan)
●	Ultrapure water (Merck Millipore, Billerica, MA)
●	Acetonitrile (Sigma-Aldrich, St. Louis, MO)

Instruments

●	HPLC: Paradigm MS4 (Bruker-Michrom Inc., Auburn, CA)
●	Mass spectrometer : LTQ-FT (Thermo Fisher Scientific Inc., Waltham, MA)

Methods

Sample preparation

1)	Dissolve a glycoprotein (12 μg) in 50 μL of 0.5 M Tris-HCl (pH 8.6) containing 8 M guanidine hydrochloride, and incubate it with 2 μL of 1 M dithiothreitol at 65°C for 30 min.	Comment 0

2)	Add 4.8 μL of 1 M sodium monoiodoacetate, and incubate the mixture at 25°C for 40 min in the dark.	Comment 0

3)	Remove the excess reagent by gel filtration with Sephadex G-25 (e. g., PD-10 column, GE Healthcare, Little Chalfont, UK), and lyophilize the eluate.	Comment 0

4)	Dissolve the glycoprotein in 50 μL of 50 μM phosphate buffer (pH 8.0)/10 mM EDTA, and incubate the sample with 5 units of N-Glycosidase F at 37°C for 24 h.	Comment 0

5)	Add cold ethanol (final concentration: 60%) to precipitate the deglycosylated protein, and incubate it −20°C for 2 h.	Comment 0

6)	Centrifuge the sample at 15,000 × g for 15 min, and dry the supernatant by SpeedVac.	Comment 0

7)	Dissolve the oligosaccharides in 250 μL of 0.5 M NaBH₄, and incubate it at 25°C for 16 h.	Comment 0

8)	Terminate the reaction with diluted acetic acid and, extract the borohydrate-reduced oligosaccharides with a solid-phase tip (e. g. an ENVI carb C cartrige, Supelco/Sigma-Aldrich, Bellefonte, PA).	Comment 0

Dry the eluate, and dissolve the borohydrate-reduced oligosaccharides in water (30 μL).

Comment 0

LC/MS

1)	Equilibrate a graphitized carbon column (e. g. HyperCarb 0.075 – 0.2 mM i. d. × 150 mM, 5μM, Thermo Fisher Scientific, Waltham, MA, USA) with 95% of mobile phase A at an appropriate flow rate (0.2–3 mL/min) for 20–30 min. Mobile phase A: 5 mM NH₄HCO₃/ 2% acetonitrile; mobile phase B: 5 mM NH₄HCO₃/ 80% acetonitrile	Comment 0

2)	Attach the nanospray tip to the outlet of the column, and set it on the x-y-z translational stage (AMR Inc., Tokyo, Japan).	Comment 0

3)	Apply a voltage of 2.5 kV to the tip, and check the stable spray with the mobile phase.	Comment 0

Pass a sample (2 μL) through the cartridge into a LC injector valve, and start simultaneously both the chromatography gradient and mass spectrometer data collection. Acquire mass spectra at a range of m/z 800–2,000 and MS/MS spectra in a data-dependent manner. Gradient: 8−50% of mobile phase B in 60 min, linear. Collision energy: 25%

Comment 0

Initial amount

5 μg protein

Figure & Legends

(A), Total ion current chromatogram of N-glycans released from recombinant human follicle stimulating hormone (FSH).

(B), A representative mass spectrum acquired at 25.4 min (peak *).

(C), Product ion spectrum acquired from m/z 1113.4 as a precursor.

LC: HPLC, Paradigm MS4 (Bruker-Michrom Inc., Auburn, CA); column, 0.1 × 150 mm; flow rate, 500 nL/min. MS: mass spectrometer, LTQ-FT (Thermo Fisher Scientific Inc.); nano-ESI probe, (AMR Inc.)

Copyrights

Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses

Date of registration：2015-05-08 15:07:08