Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA207513

Local Sample ID:	14
Subject ID:	SU002248
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002248
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
14	SA207513	FL025444	KO	Factor

Collection:

Collection ID:	CO002241
Collection Summary:	Extraction of mouse retina lipids was carried out using a biphasic solvent system of cold methanol, methyl tert-butyl ether (MTBE), and PBS using the extraction method by Matyash et al. (J Lipid Res 2008, 49, (5), 1137-46). In a randomized sequence, tissue lipids (~10 mg) were extracted in bead-mill tubes (ceramic 1.4 mm, Mo-Bio, Qiagen, Germantown, MD) containing a solution of 225 mL MeOH, 750 mL MTBE, and internal standards (Lipid standard Mouse SPLASH LipidoMix at 10 mL per sample, Avanti Polar Lipids, Alabaster, AL). Samples were homogenized in one 30 second cycle and rested on ice for 1 hour with occasional vortexing. Then, 188 mL of PBS was added followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, and the upper phases were collected. Another aliquot of 750 mL MTBE was added to the bottom aqueous layer followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, the upper phases were combined and evaporated to dryness under speedvac. Lipid extracts were reconstituted in 250 mL of mobile phase B and transferred to an LC-MS vial for analysis. Concurrently, a process blank sample was prepared and then a pooled quality control (QC) sample was prepared by taking equal volumes (~50 mL) from each sample after final resuspension.
Sample Type:	Retina

Treatment:

Treatment ID:	TR002260
Treatment Summary:	The retinas from 16 Cfap418 knockout and 16 heterozygous litter mates were collected at postnatal day 10. The retinas were kept at -80 degree before lipid extraction.

Sample Preparation:

Sampleprep ID:	SP002254
Sampleprep Summary:	Extraction of mouse retina lipids was carried out using a biphasic solvent system of cold methanol, methyl tert-butyl ether (MTBE), and PBS using the extraction method by Matyash et al. (J Lipid Res 2008, 49, (5), 1137-46). In a randomized sequence, tissue lipids (~10 mg) were extracted in bead-mill tubes (ceramic 1.4 mm, Mo-Bio, Qiagen, Germantown, MD) containing a solution of 225 mL MeOH, 750 mL MTBE, and internal standards (Lipid standard Mouse SPLASH LipidoMix at 10 mL per sample, Avanti Polar Lipids, Alabaster, AL). Samples were homogenized in one 30 second cycle and rested on ice for 1 hour with occasional vortexing. Then, 188 mL of PBS was added followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, and the upper phases were collected. Another aliquot of 750 mL MTBE was added to the bottom aqueous layer followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, the upper phases were combined and evaporated to dryness under speedvac. Lipid extracts were reconstituted in 250 mL of mobile phase B and transferred to an LC-MS vial for analysis. Concurrently, a process blank sample was prepared and then a pooled quality control (QC) sample was prepared by taking equal volumes (~50 mL) from each sample after final resuspension. Injection volumes of 2 uL for positive and 10 uL for negative mode, and iterative, tandem mass spectrometry was conducted using the same LC gradient at collision energies of 20 V and 27.5 V, respectively.

Sampleprep ID:

SP002254

Sampleprep Summary:

Extraction of mouse retina lipids was carried out using a biphasic solvent system of cold methanol, methyl tert-butyl ether (MTBE), and PBS using the extraction method by Matyash et al. (J Lipid Res 2008, 49, (5), 1137-46). In a randomized sequence, tissue lipids (~10 mg) were extracted in bead-mill tubes (ceramic 1.4 mm, Mo-Bio, Qiagen, Germantown, MD) containing a solution of 225 mL MeOH, 750 mL MTBE, and internal standards (Lipid standard Mouse SPLASH LipidoMix at 10 mL per sample, Avanti Polar Lipids, Alabaster, AL). Samples were homogenized in one 30 second cycle and rested on ice for 1 hour with occasional vortexing. Then, 188 mL of PBS was added followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, and the upper phases were collected. Another aliquot of 750 mL MTBE was added to the bottom aqueous layer followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, the upper phases were combined and evaporated to dryness under speedvac. Lipid extracts were reconstituted in 250 mL of mobile phase B and transferred to an LC-MS vial for analysis. Concurrently, a process blank sample was prepared and then a pooled quality control (QC) sample was prepared by taking equal volumes (~50 mL) from each sample after final resuspension. Injection volumes of 2 uL for positive and 10 uL for negative mode, and iterative, tandem mass spectrometry was conducted using the same LC gradient at collision energies of 20 V and 27.5 V, respectively.

Combined analysis:

Analysis ID	AN003542	AN003543
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 6550	Agilent 6550
Column	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6540 QTOF	Agilent 6545 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	pmol per sample	pmol per sample

Chromatography:

Chromatography ID:	CH002616
Chromatography Summary:	Positive Mode RP LCMS
Instrument Name:	Agilent 6550
Column Name:	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	65 C
Flow Gradient:	The chromatography gradient for both positive and negative modes started at 15% mobile phase B then increased to 30% B over 2.4 min. It sequentially increased to 48% B from 2.4 – 3.0 min, 82% B from 3 – 13.2 min, and 99% B from 13.2 – 13.8 min where it’s held until 16.7 min and returned to the initial conditions and equilibrated for 5 min.
Flow Rate:	0.4 mL min
Solvent A:	40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	90% isopropanol/9% acetonitrile/1% water;0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	Reversed phase

Chromatography ID:	CH002617
Chromatography Summary:	Negative Mode RP LCMS
Instrument Name:	Agilent 6550
Column Name:	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	65 C
Flow Gradient:	The chromatography gradient for both positive and negative modes started at 15% mobile phase B then increased to 30% B over 2.4 min. It sequentially increased to 48% B from 2.4 – 3.0 min, 82% B from 3 – 13.2 min, and 99% B from 13.2 – 13.8 min where it’s held until 16.7 min and returned to the initial conditions and equilibrated for 5 min.
Flow Rate:	0.4 mL min
Solvent A:	40% water/60% acetonitrile; 10 mM ammonium formate
Solvent B:	90% isopropanol/9% acetonitrile/1% water; 10 mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003300
Analysis ID:	AN003542
Instrument Name:	Agilent 6540 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	For positive mode, the source gas temperature was set to 225 °C, with a drying gas flow of 11 L/minute, nebulizer pressure of 40 psig, sheath gas temp of 350 °C and sheath gas flow of 11 L/minute. VCap voltage is set at 3500 V, nozzle voltage 500V, fragmentor at 110 V, skimmer at 85 V and octopole RF peak at 750 V. For data processing, Agilent MassHunter (MH) Workstation and software packages MH Qualitiative and MH Quantitative were used. The pooled QC (n=8) and process blank (n=4) were injected throughout the sample queue to ensure the reliability of acquired lipidomics data. For lipid annotation, accurate mass and MS/MS matching was used with the Agilent Lipid Annotator library. Results from the positive and negative ionization modes from Lipid Annotator were merged based on the class of lipid identified. Data exported from MH Quantitative was evaluated using Excel where initial lipid targets are parsed based on the following criteria. Only lipids with relative standard deviations (RSD) less than 30% in QC samples are used for data analysis. Additionally, only lipids with background AUC counts in process blanks that are less than 30% of QC are used for data analysis. The parsed excel data tables are normalized based on the ratio to class-specific internal standards, then to sum prior to statistical analysis.
Ion Mode:	POSITIVE

MS ID:	MS003301
Analysis ID:	AN003543
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	For negative mode, the source gas temperature was set to 300 °C, with a drying gas flow of 11 L/minute, a nebulizer pressure of 30 psig, sheath gas temp of 350 °C and sheath gas flow 11 L/minute. VCap voltage was set at 3500 V, nozzle voltage 75 V, fragmentor at 175 V, skimmer at 75 V and octopole RF peak at 750 V. For data processing, Agilent MassHunter (MH) Workstation and software packages MH Qualitiative and MH Quantitative were used. The pooled QC (n=8) and process blank (n=4) were injected throughout the sample queue to ensure the reliability of acquired lipidomics data. For lipid annotation, accurate mass and MS/MS matching was used with the Agilent Lipid Annotator library. Results from the positive and negative ionization modes from Lipid Annotator were merged based on the class of lipid identified. Data exported from MH Quantitative was evaluated using Excel where initial lipid targets are parsed based on the following criteria. Only lipids with relative standard deviations (RSD) less than 30% in QC samples are used for data analysis. Additionally, only lipids with background AUC counts in process blanks that are less than 30% of QC are used for data analysis. The parsed excel data tables are normalized based on the ratio to class-specific internal standards, then to sum prior to statistical analysis.
Ion Mode:	NEGATIVE