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MB Sample ID: SA176083
| Local Sample ID: | Sample_p_QC5 |
| Subject ID: | SU001975 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001975 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Sample_p_QC5 | SA176083 | FL021836 | QC | group |
Collection:
| Collection ID: | CO001968 |
| Collection Summary: | Overnight fasted mice were euthanized with avertin after light onset. Five replicates each of both Retinal and RPE tissues (from wild type and knock out mice) were harvested/dissected, flash frozen and stored in -80 ºC until extraction. Tissues were thawed on ice and extracted using methanol:chloroform (2:1) at 4C and phase separated using water. Top aq. phase was recovered as metabolite mixture and dried overnight in speedVac and reconstituted in 100 µL of 0.1% Formic acid in water. Ten µl from each tube was removed to create a pooled QC sample that was injected at the beginning and end of the MS sequence run and additional QC samples were injected after every 5 sample injection. |
| Sample Type: | Eye tissue |
Treatment:
| Treatment ID: | TR001987 |
| Treatment Summary: | NA |
Sample Preparation:
| Sampleprep ID: | SP001981 |
| Sampleprep Summary: | Overnight fasted mice were euthanized with avertin after light onset. Five replicates each of both Retinal and RPE tissues (from wild type and knock out mice) were harvested/dissected, flash frozen and stored in -80 ºC until extraction. Tissues were thawed on ice and extracted using methanol:chloroform (2:1) at 4C and phase separated using water. Top aq. phase was recovered as metabolite mixture and dried overnight in speedVac and reconstituted in 100 µL of 0.1% Formic acid in water. Ten µl from each tube was removed to create a pooled QC sample that was injected at the beginning and end of the MS sequence run and additional QC samples were injected after every 5 sample injection. |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003078 | AN003079 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters Acquity BEH C18 (100 x 2mm,1.7um) | Waters Acquity BEH C18 (100 x 2mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Orbitrap IDX | Orbitrap IDX |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | intensity | intensity |
Chromatography:
| Chromatography ID: | CH002276 |
| Chromatography Summary: | Low pH polar |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters Acquity BEH C18 (100 x 2mm,1.7um) |
| Column Temperature: | 30C |
| Flow Gradient: | 0-8 minutes 50% B, 8 – 13 minutes held at 98% B, 13.1 to 15.0 minutes revert to 0% B to re-equilibration for next injection |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002863 |
| Analysis ID: | AN003078 |
| Instrument Name: | Orbitrap IDX |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Conventional DDA mode was used for data acquisition and samples were randomized during extraction and analysis on the mass spectrometer. The injection volume was 5 µL. ESI source condition were set as follows: Ion source type: H-ESI; positive ion voltage set at 3500 and negative at 2500. Sheath gas was set at 35 and auxiliary gas at 7. Ion transfer tube temperature was set at 275 °C while the vaporizer temperature was set at 320 °C. Data was acquired in full MS mode (1 µscan) at a resolution of 120,000 with a scan range of 67-1000 m/z at a normalized AGC Target of 25% and 50 millisecond of maximum injection time was allowed. RF lens amplitude was set at 35%. Tandem MS/MS performed by applying quadrupole isolation with an isolation window of 1.6 m/z. Activation type was set at HCD and masses were fragmented with HCD Assisted Collision Energy (%) of 15,35,50. Fragment masses were detected by Orbitrap at a resolution of 30,000. For data analysis, Thermo *.RAW files were converted to open source *.mzML format using msConvert software (http://proteowizard.sourceforge.net/download.html) and brought into MS-DIAL software for analysis (http://prime.psc.riken.jp/compms/msdial/main.html). MS-DIAL analysis parameters were as follows: MS1 tolerance was set at 0.01 Da and Ms2 tolerance at 0.025 Da. Data was collected from 0-15 min at a mass range from 50-1000 Da. Peak detection was performed with a minimum peak height of 1x105 and mass slice width of 0.1 Da. Metabolites were identified by searching the MS2 spectra in MS-DIAL public database (290,915 records for positive and 36,848 entries for negative mode; Version 14) using a mass tolerance of 0.01 Da for MS1 and 0.05 Da for MS/MS with identification score cutoff of 60%. Additionally, peaks were also searched against in-house IROA library (in both positive and negative mode) with a mass tolerance of 0.01, identification cutoff of 90% and RT tolerance of 0.5 min (Mass Spectrometry Metabolite Library of Standards (MSMLS); http://iroatech.com/). For peak alignment maximum retention tolerance was set at 0.2 min. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002864 |
| Analysis ID: | AN003079 |
| Instrument Name: | Orbitrap IDX |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Conventional DDA mode was used for data acquisition and samples were randomized during extraction and analysis on the mass spectrometer. The injection volume was 5 µL. ESI source condition were set as follows: Ion source type: H-ESI; positive ion voltage set at 3500 and negative at 2500. Sheath gas was set at 35 and auxiliary gas at 7. Ion transfer tube temperature was set at 275 °C while the vaporizer temperature was set at 320 °C. Data was acquired in full MS mode (1 µscan) at a resolution of 120,000 with a scan range of 67-1000 m/z at a normalized AGC Target of 25% and 50 millisecond of maximum injection time was allowed. RF lens amplitude was set at 35%. Tandem MS/MS performed by applying quadrupole isolation with an isolation window of 1.6 m/z. Activation type was set at HCD and masses were fragmented with HCD Assisted Collision Energy (%) of 15,35,50. Fragment masses were detected by Orbitrap at a resolution of 30,000. For data analysis, Thermo *.RAW files were converted to open source *.mzML format using msConvert software (http://proteowizard.sourceforge.net/download.html) and brought into MS-DIAL software for analysis (http://prime.psc.riken.jp/compms/msdial/main.html). MS-DIAL analysis parameters were as follows: MS1 tolerance was set at 0.01 Da and Ms2 tolerance at 0.025 Da. Data was collected from 0-15 min at a mass range from 50-1000 Da. Peak detection was performed with a minimum peak height of 1x105 and mass slice width of 0.1 Da. Metabolites were identified by searching the MS2 spectra in MS-DIAL public database (290,915 records for positive and 36,848 entries for negative mode; Version 14) using a mass tolerance of 0.01 Da for MS1 and 0.05 Da for MS/MS with identification score cutoff of 60%. Additionally, peaks were also searched against in-house IROA library (in both positive and negative mode) with a mass tolerance of 0.01, identification cutoff of 90% and RT tolerance of 0.5 min (Mass Spectrometry Metabolite Library of Standards (MSMLS); http://iroatech.com/). For peak alignment maximum retention tolerance was set at 0.2 min. |
| Ion Mode: | NEGATIVE |
