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MB Sample ID: SA207516
Local Sample ID: | 1 |
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 |
---|---|---|---|---|
1 | SA207516 | 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. |
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 |