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Simultaneous determination of intracellular nucleotide sugars

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Introduction Protocol References Credit lines
Category
Isolation & structural analysis of glycans
Protocol Name

Simultaneous determination of intracellular nucleotide sugars

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

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

Ion-pair reverse phase HPLC

nucleotide sugars

Reagents
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Ice-cold ethanol

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PBS

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Acetonitrile

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100 mM triethylamine acetate buffer (pH 7.0)

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0.1% trifluoroacetic acid

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10 mM NH4HCO3

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

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

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BCA assay kit (Pierce)

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

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Standard nucleotide sugars

Instruments
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Handy Sonic Disruptor (Tomy Seiko Co. Ltd.)
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Speed Vac Concentrator (Labconco)
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Prominence HPLC system equipped with a UV detector (Shimadzu)
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Envi-Carb column (1 ml/100 mg; Supelco Inc.)
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Inertsil ODS-4 or Inertsil ODS-3 (particle size: 3 μm; 150 × 4.6 mm; GL Science)
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Inertsil ODS guard column (particle size: 3 μm; 10 × 4.6 mm; GL Science)
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(Option) Inertsil ODS (particle size: 3 μm; 150 × 2.1 mm; GL Science)
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Esquire HCT ESI-MS (Bruker Daltoniks)
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Agilent 1100 series HPLC system (Agilent Technologies)
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

 

Figure 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.

Figure 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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