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Simultaneous determination of intracellular nucleotide sugars
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Simultaneous determination of intracellular nucleotide sugars

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Category
Isolation & structural analysis of glycans
Protocol Name

Simultaneous determination of intracellular nucleotide sugars

Authors
Nakajima, Kazuki *
Systems Glycobiology Research Group, Chemical Biology Department, RIKEN Advanced Science Institute

Taniguchi, Naoyuki
Systems Glycobiology Research Group, Chemical Biology Department, RIKEN Advanced Science Institute
*To whom correspondence should be addressed.
KeyWords
Reagents

Ice-cold ethanol

PBS

Acetonitrile

100 mM triethylamine acetate buffer (pH 7.0)

0.1% trifluoroacetic acid

10 mM NH4HCO3

Buffer A for HPLC analysis: 100 mM potassium phosphate buffer (pH 6.4) supplemented with 8 mM tetrabutylammonium hydrogensulphate

Buffer B for HPLC analysis: 80% buffer A plus 20% acetonitrile

BCA assay kit (Pierce, Rockford, IL)

(Option) 20 mM triethylamine acetate buffer (pH 6.0)

Standard nucleotide sugars

Instruments

Handy Sonic Disruptor (Tomy Seiko Co. Ltd., Tokyo, Japan)

Speed Vac Concentrator (Labconco Co., Kansas City, MO)

Prominence HPLC system equipped with a UV detector (Shimadzu Corp., Kyoto, Japan)

Envi-Carb column (1 mL/100 mg; Supelco/Sigma-Aldrich, Bellefonte, PA)

Inertsil ODS-4 or Inertsil ODS-3 (particle size: 3 μm; 150 × 4.6 mm; GL Science, Tokyo, Japan)

Inertsil ODS guard column (particle size: 3 μm; 10 × 4.6 mm; GL Science)

(Option) Inertsil ODS (particle size: 3 μm; 150 × 2.1 mm; GL Science)

Esquire HCT ESI-MS (Bruker Daltonics Inc., Billerica, MA)

Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA)

Methods
1.

Preparation of cellular nucleotide sugars

1) 

 Prepare subconfluent cells in 6-well plates.

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2) 

 Wash the cells on the culture dishes with cold PBS and quickly collect into 2-mL tubes with 500 μL of cold PBS.

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3) 

 Add 1.5 mL of ice-cold ethanol and lyse with the sonicator.

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4) 

 Spike with unnatural GDP-Glc (2.5 nmol) to normalize recovery in the preparation.

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5) 

 Centrifuge at 16,000 × g for 15 min at 4°C to remove insoluble materials.

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6) 

 Lyophilize the supernatants with the speed vac concentrator.

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7) 

 Dissolve the freeze-dried samples in 1 mL of 10 mM NH4HCO3.

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8) 

 Apply the sample solutions to the Envi-Carb column briefly conditioned with 80% acetonitrile in 0.1% trifluoroacetic acid (2 mL) followed by 1 mL of water.

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9) 

 Sequentially wash the column with 1 mL of water, 1 mL of 25% acetonitrile, 200 μL of water and 1 mL of 50 mM triethylamine acetate buffer (pH 7).

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10) 

 Elute the nucleotide sugars with 1 mL of 25% acetonitrile in 50 mM triethylamine acetate buffer (pH 7).

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11) 

 Lyophilize the eluates.

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2.

Ion-pair reversed-phase HPLC

1) 

 Dissolve the extracted nucleotide sugars in 50 μL of water.

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2) 

 Analyze aliquots (20 μL) of the nucleotide sugar solutions by HPLC at 40°C on the Inertsil ODS-3 or Inertsil ODS-4 in combination with the guard column. Maintain the flow rate at 1 mL/min. Detect the nucleotide sugars at 254 nm. Elute the samples using the following gradient profile: 100% (v/v) buffer A for 13 min; 0–77% (v/v) linear gradient of buffer B for 22 min; 77–100% (v/v) buffer B for 1 min; and 100% buffer B for 14 min.

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3) 

 Quantify the nucleotide sugar levels based on the peak areas of calibration curves with the standard nucleotide sugars.

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4) 

 Normalize the nucleotide sugar levels by the recovery of exogenous GDP-Glc, and then express the levels as pmol/mg protein. Determine the protein contents in the cells using the BCA assay kit.

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3.

Ion-pair reversed-phase HPLC ESI-MS/MS

1) 

 Analyze the same samples by ion-pair reversed-phase LC ESI-MS/MS at 40°C on the Inertsil ODS-4 (150 × 2.1 mm). Maintain the flow rate at 0.2 mL/min. The optimized elution gradient is as follows: 100% buffer A for 15 min; 0–20% linear gradient of buffer B for 20 min; 20–100% linear gradient of buffer B for 1 min; and 100% buffer B for 14 min. Buffer B is 80% buffer A plus 20% acetonitrile.

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2) 

 Introduce continuously the eluate into an ESI source, and ionize nucleotide sugars in negative ion mode. Mass spectrometry is operated using the multiple reaction-monitoring mode for the number of the predetermined mass. For the MS/MS run, a continuous flow of helium gas is used for collisional cooling.

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3) 

 Quantify the nucleotide sugar levels based on the peak area in extraction ion chromatograms of the major fragment ions as follows: m/z 613→322 for CMP-NeuAc, m/z 565→323 for UDP-Gal and UDP-Glc, m/z 606→385 for UDP-HexNAc, m/z 604→442 for GDP-Man, m/z 579→323 for UDP-GlcA, and m/z 588→442 for GDP-Fuc.

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Notes
  • Prepare the HPLC buffer carefully, especially with regard to the pH value adjustment, because the pH of the buffer affects the separation of individual molecules.
  • Use of the Inertsil ODS-4 and Inertsil ODS-3 is crucial for achieving the separation of nucleotide sugars, particularly that of UDP-GlcNAc and UDP-GalNAc. These columns are end-capped reversed-phase columns with higher hydrophobicity (surface area: 450 m2/g) than a conventional column.
  • Since we cannot rule out the possibility that some of the peaks we observed contained certain contaminants in various samples, we recommend additional analysis of the same samples by LC-ESI-MS/MS (Method 3).
Figure & Legends

Figure & Legends

 

Fig. A.  Nucleotide sugars derived from Chinese hamster ovary (CHO) cells (1 × 106 cells).

Nucleotide sugars were separated by ion-pair reversed-phase HPLC. Each peak was identified by comparison with the retention times of a standard mixture. The nucleotide sugar levels were determined based on the peak areas of calibration curves and normalized to units of pmol/mg protein.

Fig. B.  Series of standard nucleotide sugars and nucleotides (500 pmol of each molecule).

 

 

These figures were originally published in Glycobiology. 20(7):865–871. 2010. “Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC” Nakajima K, Taniguchi N, et al. Oxford University Press.

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