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MB Sample ID: SA076876
| Local Sample ID: | AG130814_50 |
| Subject ID: | SU001170 |
| Subject Type: | Chemical |
| Subject Species: | None |
| Subject Comments: | Primary standards of nucleotide triphosphates (ATP, GTP, UTP and CTP). |
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Subject:
| Subject ID: | SU001170 |
| Subject Type: | Chemical |
| Subject Species: | None |
| Subject Comments: | Primary standards of nucleotide triphosphates (ATP, GTP, UTP and CTP). |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| AG130814_50 | SA076876 | FL011767 | 3 | Time (mins) |
| AG130814_50 | SA076876 | FL011767 | ATP | Nucleotide |
| AG130814_50 | SA076876 | FL011767 | Yes | Yeast Matrix |
Collection:
| Collection ID: | CO001164 |
| Collection Summary: | None |
Treatment:
| Treatment ID: | TR001184 |
| Treatment Summary: | Pure solutions of nucleotide triphosphates (ATP, GTP, CTP and UTP) were incubated under boiling ethanol conditions (95°C) during 0, 5, 10, 20, 30, 60, 120, 180, 240 and 300 minutes. |
| Treatment: | Abiotic |
Sample Preparation:
| Sampleprep ID: | SP001177 |
| Sampleprep Summary: | Tubes containing 496 µL of 75% (aq) ethanol were preheated at 95°C for 5 min, followed by the addition of 4 µL of each nucleotide standard solution (500 µM) and vigorous mixing. 4 µL of solutions “A” to “H” were added to the reaction mixture and the samples were incubated at 95°C under shaking for 0 and 15 min. Reactions were stopped by snap-freezing in liquid nitrogen and samples were stored on dry ice. Subsequently, samples were thawed and 20 µL of the 13C15N-labeled internal standard solution were added for quantitative analysis. Excess solvent was evaporated under a stream of nitrogen without heating. Finally, samples were reconstituted in 200 µL of acetonitrile-water 70:30 and stored at –40°C until analysis by LC-MS. After the experimental procedure the final concentration of all components in solutions “A” to “H” was 10 µM. Compounds to be tested were divided into eight groups containing approximately ten compounds each, mainly comprising major central carbon metabolites including amino acids, organic acids, sugar phosphates, coenzymes, etc (Table 1). |
| Processing Method: | Preparation of reagents, dilution, solvent removal under a stream of nitrogen without heating and reconstitution |
| Extraction Method: | Boiling ethanol (95°C) |
| Extract Enrichment: | Evaporation of excess of solvent under a stream of nitrogen without heating. |
| Extract Storage: | -40C |
| Sample Resuspension: | 200 µL of acetonitrile-water 70:30 |
Combined analysis:
| Analysis ID | AN001822 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Waters Acquity |
| Column | Phenomenex Luna NH2 (100 x 2.0mm,3um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Waters Synapt G2 Si QTOF |
| Ion Mode | NEGATIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH001291 |
| Chromatography Summary: | Untargeted HILIC Method |
| Instrument Name: | Waters Acquity |
| Column Name: | Phenomenex Luna NH2 (100 x 2.0mm,3um) |
| Column Temperature: | 20C |
| Flow Gradient: | 30% eluent A to 99% eluent A in 8 min, followed by isocratic elution at 99% eluent A until 14 min. A conditioning cycle of 6 min with the initial proportions of eluents A and B was performed prior to the next analysis. |
| Flow Rate: | 0.25 mL/min |
| Sample Injection: | 10 µL |
| Solvent A: | 100% water; 5 mM ammonium acetate, pH 9.9 |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 20 min |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS001685 |
| Analysis ID: | AN001822 |
| Instrument Name: | Waters Synapt G2 Si QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
| Capillary Voltage: | 2KV |
| Collision Energy: | 2 V for the low-collision energy scan, and 10–30 V for the high-collision energy scan |
| Dry Gas Flow: | Argon |
| Source Temperature: | 150C |
| Desolvation Temperature: | 400C |
| Scan Range Moverz: | 50 to 1200 Da |
