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Activated Nucleotide Sugar
Analyzing UDP Glucose
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printable Application Sheet
Notes: Activated nucleotide sugars, such as UDP-glucose [UDP-Glc] are donor substrates of glycosyltransferases involved in
protein glycosylation processes. Therefore, their availability can influence the glycosylation of proteins.
The HPLC method developed can also be used to quantify UDP-Glc, an important metabolic intermediate and the substrate of
enzymes catalyzing glycosylation reactions. Thus several enzymes producing UDP-Glc can be directly assayed using the HPLC
method described in this application note.
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Uridine 5-diphosphate (UDP)–glucose
Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-15P-2 |
| Dimensions |
2.1 x 150 mm |
| Solvents |
A: DI water + 0.1% ammonium formate pH 7.2
B: 90% acetonitrile + 10% DI water + 0.1% ammonium formate pH 6 |
| Mobile Phase |
Gradient:
| Time |
%B |
Time |
%B |
| 0.0 |
100 |
7.0 |
70 |
| 3.0 |
95 |
8.0 |
50 |
| 6.0 |
70 |
9.0 |
50 |
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| Post Time |
5 min. |
| Flow Rate |
300 µL/min. |
| Compounds |
1. UDP glucose - the monitored MRM transitions were m/z 565 to m/z 323
2. UDP hexanolamine (internal standard) - the monitored MRM transitions were m/z 502 to m/z 258
(MRM – multiple reaction monitoring in LC/MS/MS) |
| Detection |
ESI – neg - Agilent 6410 Triple Quadrupole LC/MS |
Discussion
The Aqueous Normal Phase (ANP) inverse gradient method shown above was used to analyze UDP glucose. UDP hexanolamine was used as an
internal standard. The method is rapid and simple and it can be used in measuring the metabolite. The advantages of using this method
to assay nucleotide sugars are the short separation time, excellent long term stability and rapid equilibration time when a gradient
is used.
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