Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA325775

Local Sample ID:	K562_100nM_Timecourse_Day2_Porphyrin_3
Subject ID:	SU003103
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Strain Details:	K-562

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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_Day2_Porphyrin_3	SA325775	FL038743	Day2	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.

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