Return to study ST002990 main page
MB Sample ID: SA325783
Local Sample ID: | K562_100nM_Timecourse_Day6_Porphyrin_2 |
Subject ID: | SU003103 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Cell Strain Details: | K-562 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003103 |
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_100nM_Timecourse_Day6_Porphyrin_2 | SA325783 | FL038745 | Day6 | 100nM_Folic_Acid_Growth |
Collection:
Collection ID: | CO003096 |
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: | TR003112 |
Treatment Summary: | K562 cells were cultured for 0, 1, 2, 4, or 6 days in 100 nM folic acid containing RPMI media. |
Sample Preparation:
Sampleprep ID: | SP003109 |
Sampleprep Summary: | One million cells from culture were collected via centrifugation, washed with 0.9% NaCl, and resuspended in 150 µl of porphyrin extraction buffer (1:4 ratio of 1.7 M HCl:ACN, 1µM deuteroporphyrin IX (Frontier Scientific, D510-9)) and 0.5 µM isotopically labeled amino acids (Cambridge Isotopes, MSK-A2-1.2)). Samples were vigorously shaken for 20 min at 16ºC in a thermomixer (Eppendorf), sonicated for 10 cycles at 4ºC with 30 sec on and 30 sec off, then incubated at 4ºC for 10 min. Following incubation on ice, samples were centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was collected and 40.5 µl super-saturated MgSO4 and 12 µl 5 M NaCl were added. Samples were vortexed for 30 sec and further shaken for 10 min at 16 ºC in a thermomixer. Finally, a 10 min 10,000 rpm centrifugation was used to separate the organic layer (upper) from the aqueous layer (lower). The upper organic layer was collected. |
Combined analysis:
Analysis ID | AN004910 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Thermo Vanquish |
Column | Phenomenex Kinetex C18 (150 x 3mm,2.6um) |
MS Type | ESI |
MS instrument type | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap |
Ion Mode | POSITIVE |
Units | Arbitrary Units |
Chromatography:
Chromatography ID: | CH003705 |
Chromatography Summary: | Sample (5 uL) was injected onto a 2.6 μm, 150 x 3 mm C18 column (Phenomenex, 00F-4462-Y0) equipped with a 3.0 mm safe-guard column (Phenomenex, AJ0-8775). Column compartment was heated to 45 ºC. Porphyrins were separated with a chromatographic gradient at a flow rate of 0.800 ml min−1 as follows: 0–2 min: 5% B; 2-19min: linear gradient from 5% to 95% B; 19-21min: 95% B; 21.1-23min: return to 5% B. |
Instrument Name: | Thermo Vanquish |
Column Name: | Phenomenex Kinetex C18 (150 x 3mm,2.6um) |
Column Temperature: | 45 |
Flow Gradient: | linear gradient from 5%B to 95%B |
Flow Rate: | 0.8 mL/min |
Solvent A: | 95% water/5% acetonitrile; 0.1% formic acid |
Solvent B: | 5% water/95% acetonitrile; 0.1% formic acid |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS004653 |
Analysis ID: | AN004910 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The mass spectrometer was operated in full-scan, positive ionization mode using a narrow-range scan: 450-700m/z, with an additional tSIM scan for hemin (616.1767 m/z), CoproP (655.2762 m/z), and PPIX (563.2653 m/z). 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: | POSITIVE |