Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA169026

Local Sample ID:	LPIAT1 KO #2-7
Subject ID:	SU001894
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	8 weeks old
Gender:	Male

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Subject:

Subject ID:	SU001894
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	8 weeks old
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
LPIAT1 KO #2-7	SA169026	FL020161	LPIAT1 KO #2	Tissue

Collection:

Collection ID:	CO001887
Collection Summary:	MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. 1 × 106 cells were seeded on cell culture dishes (6.0 cm diameter) and the next day, cells were washed twice with PBS and collected to a safe-lock poly-propylene tube (2 ml) with 1 M HCl (500 μl), followed by centrifugation (15,000 g, 5 min at 4 ºC). Supernatants were removed rapidly and resuspended with 750 μl of quench mix, 170 μl of H2O.
Sample Type:	Fibroblasts

Treatment:

Treatment ID:	TR001907
Treatment Summary:	MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine.

Sample Preparation:

Sampleprep ID:	SP001900
Sampleprep Summary:	The following solutions were prepared for lipid extractions; the quench mixture comprising 484 ml MeOH, 242 ml CHCl3, and 23.55 ml 1 M HCl; the pre-derivatization wash composed of 240 ml CHCl3, 120 ml MeOH and 90 ml 0.01 M HCl; and the post-derivatization wash made up of 240 ml CHCl3, 120 ml MeOH, and 90 ml H2O. Wash mixtures were shaken and allowed to separate into two phases. For pre/post-derivatization wash, the upper phase of each solution was used. Cells were washed twice with PBS and collected to a safe-lock poly-propylene tube (2 ml) with 1 M HCl (500 μl), followed by centrifugation (15,000 g, 5 min at 4 ºC). Supernatants were removed rapidly and resuspended with 750 μl of quench mix, 170 μl of H2O and internal standards [10 μl containing 2 ng 12:0/13:0 PI, 17:0/20:4 PI (4)P, 17:0/20:4 PI (4,5)P2 and 17:0/20:4 PI (3,4,5)P3], followed by vortex-mixing and lipid extract steps. Lipid extraction was performed, based on procedures described by J Clark et al. The single-phase sample (a mixture of 170 μl of an aqueous sample, 2 ng of internal standards, and 750 μl of quench mix) were mixed with 725 μl of CHCl3 and 170 μl of 2M HCl, followed by vortex-mixing and centrifugation (15,000 g, 5 min at room temperature). The lower organic phase was collected into a fresh safe-lock poly-propylene tube (2 ml) and mixed with 708 μl of prederivatization wash, followed by vortex-mixing and centrifugation (15,000 g, 3 min at room temperature). The lower phase was collected into another fresh tube and subjected to derivatization. Derivatization of lipids was performed in a fume hood with adequate personal safety equipment as follows, based on procedures described by J Clark et al7. Fifty μl trimethylsilyl diazomethane in hexane (2 M solution; Sigma-Aldrich) was added to the lipid extracts (~1 ml), and after leaving to stand 10 min at room temperature, the reaction was quenched with 6 μl of acetic acid. Next, 700 μl post-derivatization wash solution was added to the mixture, followed by centrifugation (1,500 g, 3 min). The resultant lower phase was collected and rewashed with a 700 μl post-derivatization wash solution. Then 90 μl of MeOH and 10 μl of H2O were added to the final collected lower phase. The samples were dried up with a centrifugal evaporator. Finally, samples were dissolved in 80μl MeOH, sonicated briefly, and 20 μl H2O was added. To avoid degradation, the samples were stored at -80ºC until use.

Sampleprep ID:

SP001900

Sampleprep Summary:

The following solutions were prepared for lipid extractions; the quench mixture comprising 484 ml MeOH, 242 ml CHCl3, and 23.55 ml 1 M HCl; the pre-derivatization wash composed of 240 ml CHCl3, 120 ml MeOH and 90 ml 0.01 M HCl; and the post-derivatization wash made up of 240 ml CHCl3, 120 ml MeOH, and 90 ml H2O. Wash mixtures were shaken and allowed to separate into two phases. For pre/post-derivatization wash, the upper phase of each solution was used. Cells were washed twice with PBS and collected to a safe-lock poly-propylene tube (2 ml) with 1 M HCl (500 μl), followed by centrifugation (15,000 g, 5 min at 4 ºC). Supernatants were removed rapidly and resuspended with 750 μl of quench mix, 170 μl of H2O and internal standards [10 μl containing 2 ng 12:0/13:0 PI, 17:0/20:4 PI (4)P, 17:0/20:4 PI (4,5)P2 and 17:0/20:4 PI (3,4,5)P3], followed by vortex-mixing and lipid extract steps. Lipid extraction was performed, based on procedures described by J Clark et al. The single-phase sample (a mixture of 170 μl of an aqueous sample, 2 ng of internal standards, and 750 μl of quench mix) were mixed with 725 μl of CHCl3 and 170 μl of 2M HCl, followed by vortex-mixing and centrifugation (15,000 g, 5 min at room temperature). The lower organic phase was collected into a fresh safe-lock poly-propylene tube (2 ml) and mixed with 708 μl of prederivatization wash, followed by vortex-mixing and centrifugation (15,000 g, 3 min at room temperature). The lower phase was collected into another fresh tube and subjected to derivatization. Derivatization of lipids was performed in a fume hood with adequate personal safety equipment as follows, based on procedures described by J Clark et al7. Fifty μl trimethylsilyl diazomethane in hexane (2 M solution; Sigma-Aldrich) was added to the lipid extracts (~1 ml), and after leaving to stand 10 min at room temperature, the reaction was quenched with 6 μl of acetic acid. Next, 700 μl post-derivatization wash solution was added to the mixture, followed by centrifugation (1,500 g, 3 min). The resultant lower phase was collected and rewashed with a 700 μl post-derivatization wash solution. Then 90 μl of MeOH and 10 μl of H2O were added to the final collected lower phase. The samples were dried up with a centrifugal evaporator. Finally, samples were dissolved in 80μl MeOH, sonicated briefly, and 20 μl H2O was added. To avoid degradation, the samples were stored at -80ºC until use.

Combined analysis:

Analysis ID	AN002949
Analysis type	MS
Chromatography type	Unspecified
Chromatography system	Shimadzu Nexera UC
Column	ULTRON AF-HILIC-CD (250 × 2.1 mm,5.0um) Shinwa Chemicalstries
MS Type	ESI
MS instrument type	Ion trap
MS instrument name	ABI SCIEX 4500 QTrap
Ion Mode	POSITIVE
Units	pmol/1000000 cells

Chromatography:

Chromatography ID:	CH002184
Instrument Name:	Shimadzu Nexera UC
Column Name:	ULTRON AF-HILIC-CD (250 × 2.1 mm,5.0um) Shinwa Chemicalstries
Column Temperature:	4℃
Flow Gradient:	0–16 min: 5% B→20% B; 16.01–18 min: 40% B; 18.01–22 min: 5% B
Flow Rate:	1.5 ml/min
Solvent A:	supercritical carbon dioxide (SCCO2)
Solvent B:	2.5% water/97.5% methanol; 0.1% formic acid
Chromatography Type:	Unspecified

MS:

MS ID:	MS002739
Analysis ID:	AN002949
Instrument Name:	ABI SCIEX 4500 QTrap
Instrument Type:	Ion trap
MS Type:	ESI
MS Comments:	The instrument parameters of QTRAP4500 for positive ion mode were as follows: curtain gas, 20 psi; ionspray voltage, 4500 V; temperature, 300 ºC; ion source gas 1, 18 psi; ion source gas 2, 20 psi. and quantification was performed using MultiQuant™ software (SCIEX).
Ion Mode:	POSITIVE