Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA169904

Local Sample ID:	S1_0039
Subject ID:	SU001906
Subject Type:	Synthetic sample

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Subject:

Subject ID:	SU001906
Subject Type:	Synthetic sample

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
S1_0039	SA169904	FL020234	PC16;0/18;2, AAPH+hemin	Treatments
S1_0039	SA169904	FL020234	4h	Treatment times
S1_0039	SA169904	FL020234	normal air	Air conditions

Collection:

Collection ID:	CO001899
Collection Summary:	PC16:0/18:2, PC16:0/20:4, and PC16:0/22:6 were purchased from Avanti Polar Lipids (Albaster, AL). Each PC was oxidized by using several LPO systems, such as AAPH, autoxidation, AAPH+hemin and CuSO4+AsA.
Sample Type:	Standard phosphadylcholines

Treatment:

Treatment ID:	TR001919
Treatment Summary:	For in vitro experiments, a mixture containing 500 µM PC16:0/PUFAs (PC16:0/18:2, PC16:0/20:4, and PC16:0/22:6), 0.5% ethanol, and either 50 mM AAPH or 50 mM AAPH + 10 µM hemin was incubated in phosphate-buffered saline (PBS; pH 7.4) at 37 °C under normal or 18O2 air. For autoxidation, a solution of 500 nmol of PC16:0/18:2 in chloroform (1 mL) was immediately dried using nitrogen gas in a glass tube. The dried samples were incubated at 37 °C for 24 h and then resuspended in methanol (200 µL). For CuSO4 + AsA–induced peroxidation, a solution containing 500 µM PC16:0/18:2 and 500 µM CuSO4 + 1 mM AsA was incubated in PBS (pH 7.4) at 37 °C for 72 h.

Treatment ID:

TR001919

Treatment Summary:

For in vitro experiments, a mixture containing 500 µM PC16:0/PUFAs (PC16:0/18:2, PC16:0/20:4, and PC16:0/22:6), 0.5% ethanol, and either 50 mM AAPH or 50 mM AAPH + 10 µM hemin was incubated in phosphate-buffered saline (PBS; pH 7.4) at 37 °C under normal or 18O2 air. For autoxidation, a solution of 500 nmol of PC16:0/18:2 in chloroform (1 mL) was immediately dried using nitrogen gas in a glass tube. The dried samples were incubated at 37 °C for 24 h and then resuspended in methanol (200 µL). For CuSO4 + AsA–induced peroxidation, a solution containing 500 µM PC16:0/18:2 and 500 µM CuSO4 + 1 mM AsA was incubated in PBS (pH 7.4) at 37 °C for 72 h.

Sample Preparation:

Sampleprep ID:	SP001912
Sampleprep Summary:	For in vitro experiments, the reaction solutions were extracted using the modified Bligh and Dyer method. Briefly, a mixed solution of 1 mL methanol and 1 mL chloroform, containing 100 μM dibutylhydroxytoluene and ethylenediaminetetraacetic acid as antioxidants, and 100 nM PC15:0/18:1-d7 as an internal standard was added to the reaction solution. After 1 min vortex, the organic layer was collected in a glass tube. The extracted solution was dried under a stream of nitrogen gas, and the residue was dissolved in methanol (200 µL) and stored at −80 °C before injection into the HPLC column.

Sampleprep ID:

SP001912

Sampleprep Summary:

For in vitro experiments, the reaction solutions were extracted using the modified Bligh and Dyer method. Briefly, a mixed solution of 1 mL methanol and 1 mL chloroform, containing 100 μM dibutylhydroxytoluene and ethylenediaminetetraacetic acid as antioxidants, and 100 nM PC15:0/18:1-d7 as an internal standard was added to the reaction solution. After 1 min vortex, the organic layer was collected in a glass tube. The extracted solution was dried under a stream of nitrogen gas, and the residue was dissolved in methanol (200 µL) and stored at −80 °C before injection into the HPLC column.

Combined analysis:

Analysis ID	AN002968	AN002969
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Nexera LC system (Shimadzu Co., Kyoto, Japan)	Nexera LC system (Shimadzu Co., Kyoto, Japan)
Column	Inertsil ODS-P (150 x 2.1mm,3um)	Inertsil ODS-P (150 x 2.1mm,3um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Normalized amount	Normalized amount

Chromatography:

Chromatography ID:	CH002199
Instrument Name:	Nexera LC system (Shimadzu Co., Kyoto, Japan)
Column Name:	Inertsil ODS-P (150 x 2.1mm,3um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002758
Analysis ID:	AN002968
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Individual oxPCs were identified by full-scan MS/data-dependent MS/MS or parallel reaction monitoring (PRM). The ionization conditions were as follows: ionization mode = positive, sheath gas flow rate = 40 arbitrary units, auxiliary gas flow rate = 10 arbitrary units, spray voltage = 2000 V, capillary temperature = 265 °C, S-lens level = 50, and heater temperature = 425 °C. The experimental conditions for full-scan MS were as follows: resolving power = 70000, automatic gain control target = 1 × 106, trap fill time = 100 ms, scan range = m/z 120–1500. The experimental conditions for MS/MS and PRM were as follows: resolving power = 17500, automatic gain control target = 1 × 106, trap fill time = 80 ms, isolation width = ±0.6 Da, fixed first mass = m/z 80, normalized collision energy = 20 or 35 eV, intensity threshold of precursor ions for MS/MS analysis = 3100, apex trigger = 2–4 s, and dynamic exclusion = 2 s. The intensity threshold of precursor ions for MS/MS analysis and the dynamic exclusion were set to 1 × 104 and 1 s, respectively. LC/HRMS/MS analysis was controlled using Xcalibur 4.2.47 software (Thermo Fisher Scientific).
Ion Mode:	POSITIVE

MS ID:	MS002759
Analysis ID:	AN002969
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Individual oxPCs were identified by full-scan MS/data-dependent MS/MS or parallel reaction monitoring (PRM). The ionization conditions were as follows: ionization mode = negative, sheath gas flow rate = 40 arbitrary units, auxiliary gas flow rate = 10 arbitrary units, spray voltage = 2000 V, capillary temperature = 265 °C, S-lens level = 50, and heater temperature = 425 °C. The experimental conditions for full-scan MS were as follows: resolving power = 70000, automatic gain control target = 1 × 106, trap fill time = 100 ms, scan range = m/z 120–1500. The experimental conditions for MS/MS and PRM were as follows: resolving power = 17500, automatic gain control target = 1 × 106, trap fill time = 80 ms, isolation width = ±0.6 Da, fixed first mass = m/z 80, normalized collision energy = 20 or 35 eV, intensity threshold of precursor ions for MS/MS analysis = 3100, apex trigger = 2–4 s, and dynamic exclusion = 2 s. The intensity threshold of precursor ions for MS/MS analysis and the dynamic exclusion were set to 1 × 104 and 1 s, respectively. LC/HRMS/MS analysis was controlled using Xcalibur 4.2.47 software (Thermo Fisher Scientific).
Ion Mode:	NEGATIVE