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MB Sample ID: SA325830
| Local Sample ID: | K562_2000nM_FA_Unlabeled_HILIC_2 |
| Subject ID: | SU003105 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | K-562 |
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Subject:
| Subject ID: | SU003105 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | K-562 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| K562_2000nM_FA_Unlabeled_HILIC_2 | SA325830 | FL038755 | 2000nM_FA_Unlabeled | Treatment |
Collection:
| Collection ID: | CO003098 |
| Collection Summary: | One million cells from culture were collected via centrifugation for 20 seconds at 18,000xG, washed with 0.9% NaCl, and collected via centrifugation for 20 seconds at 18,000xG |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003114 |
| Treatment Summary: | Culture of K562 cells for 7 days in RPMI media containing 2000 nM folic acid or 100 nM folic acid. At day 8, media was changed to 2000 nM or 100 nM folic acid with unlabeled serine and glycine, or 2-C13-Serine and 2-C13-Glycine at RPMI levels. Amino acid tracing was performed for 24 hours. The level of serine and glycine in all conditions was 30 and 10 mg/L, respectively. |
Sample Preparation:
| Sampleprep ID: | SP003111 |
| Sampleprep Summary: | Pellet was resuspended in extraction buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000 g to pellet cell debris. The supernatant was divided into two tubes and dried on ice using a liquid nitrogen dryer. One tube of dried sample was used for polar metabolite detection. Dried samples were resuspended in 25 μL water |
Combined analysis:
| Analysis ID | AN004912 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | EMD Millipore ZIC-HILIC (100 x 2.1 mm,3.5 um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH003707 |
| Chromatography Summary: | Sample was injected into a ZIC-pHILIC 150 x 2.1 mm (5 μm particle size) column (EMD Millipore). operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separation was achieved using the following conditions: buffer A was acetonitrile; buffer B was 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water; resulting pH is around 9 without pH adjustment. Gradient conditions we used were: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | EMD Millipore ZIC-HILIC (100 x 2.1 mm,3.5 um) |
| Column Temperature: | 25 |
| Flow Gradient: | linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate |
| Flow Rate: | 150 mL/min |
| Solvent A: | 100% acetonitrile |
| Solvent B: | 100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004655 |
| Analysis ID: | AN004912 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Polar metabolites were relatively quantified while referencing an in-house library of chemical standards and using TraceFinder 4.1 (Thermo Fisher Scientific, Waltham, MA, USA), with a 5 ppm mass tolerance. |
| Ion Mode: | UNSPECIFIED |
