Analysis of intracellular nucleotide sugars

 Cultivation and harvest of cells.

Yeast cells (OD_{600 nm} = 50) or Mammalian cells (8 × 10⁷ cells)

 Add 5 mL of ice-cold 1 mL formic acid saturated with 1-butanol to the cells.

 Incubate for 1 h at 4°C.

 Centrifuge at 13,000 × g for 5 min to remove cell debris.

 Dry the supernatant by centrifuge concentrator.

 Add 100 μL of distilled water.

 Filtrate a membrane filter with a pore size of 0.45 μm (DISMIC-03CP).

 Analyze the above extracted intracellular sugar nucleotide (40 μL) by HPLC (LC-10) on a cosmosil 5C₁₈-AR-II column. The column is equilibrated with 20 mM TEAA buffer (pH 7.0) at a flow rate of 1 mL·min⁻¹. UDP-sugars are detected by UV_{260 nm} absorbance.

 The peaks of detected target sugar nucleotides are collected based on the retention times of the standards.

 The collected sugar nucleotides are dried by centrifuge concentrator and resolved in 40 μL of distilled water.

 The structures of the UDP-sugars are analyzed by ESI-MS to determine its molecular mass. Mass spectra are acquired on an Esquire 3,000-plus instrument in the negative-ion mode. Conditions for ESI-MS are as follows: 68.95 kPa nebulizer flow, 300°C nozzle temperature, and 5.0 L·min⁻¹ flow of drying gas (N₂).

 The collected sugar nucleotides are reseparated on the HPLC system (LC-10) with a Develosil RPAQUEOUS column (250 × 4.6 mm). The column is equilibrated with 20 mM TEAA buffer (pH 7.0) at a flow rate of 1 mL·min⁻¹. UDP-sugars are detected by UV_{260 nm} absorbance.

(Option)

Alternatively, the collected sugar nucleotides can be separated on the HPLC system (LC-10,) with a CarboPac PA1 anion-exchange column. The column was equilibrated with solvent A (20 mM K₂HPO₄-KHPO₄, pH 7.5) at a flow rate of 0.7 mL/min for 5 min, and analyzed isocratically with solvent B (200 mM K₂HPO₄-KHPO₄, pH 7.5) at a flow rate of 0.7 mL/min for 30 min.