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MB Sample ID: SA200803
| Local Sample ID: | b1_pool_pd1074_b1_02 |
| Subject ID: | SU002176 |
| Subject Type: | Invertebrate |
| Subject Species: | Caenorhabditis elegans |
| Taxonomy ID: | 6239 |
| Gender: | Hermaphrodite |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002176 |
| Subject Type: | Invertebrate |
| Subject Species: | Caenorhabditis elegans |
| Taxonomy ID: | 6239 |
| Gender: | Hermaphrodite |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| b1_pool_pd1074_b1_02 | SA200803 | FL024359 | - | Strain |
| b1_pool_pd1074_b1_02 | SA200803 | FL024359 | pool_pd1074 | Sample type |
Collection:
| Collection ID: | CO002169 |
| Collection Summary: | Escherichia coli (E. coli) IBAT (iterative batch average method) reference material and food source used throughout this experiment. Briefly, for each biological sample, a large-scale culture plate (LSCP) was used to generate a large mixed-stage population of worms (four to seven LSCP replicates per test strain). For each LSCP, worms were collected, population size estimated, and subsequently divided into at least 12 identical aliquots of 200,000 worms in ddH2O and flash-frozen in liquid nitrogen and stored at -80°C 8. As a quality control sample, C. elegans IBAT reference material was generated and saved in 200,000 worm aliquots. |
| Sample Type: | Worms |
| Volumeoramount Collected: | 200,000 worms |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002188 |
| Treatment Summary: | No treatments. |
Sample Preparation:
| Sampleprep ID: | SP002182 |
| Sampleprep Summary: | Frozen aliquots of 200,000 C. elegans worms were retrieved from -80°C and lyophilized in a VirTis® BenchTop™ “K” Series Freeze Dryer (SP Industries, Inc.). After lyophilization, each aliquot was weighed and stored at -80°C until homogenization. Aliquots were homogenized for three minutes in a Qiagen Tissuelyser 2, using glass beads. Homogenized worms were extracted with 1.5 mL of isopropanol (IPA) at -20°C overnight (approximately 12 hours), then pelleted and the supernatant transferred to separate 2 mL centrifuge tubes. Supernatants were then dried down to completion in a Labconco Centrivap and stored at -80°C for non-polar LC-MS analysis. The pellet was extracted a second time using 80:20 methanol:water (MeOH:H2O) (v:v) for 20 minutes at room temperature (RT) while shaking at 1500 rpm. Samples were again pelleted to separate proteins, and the supernatant was transferred to separate 2 mL centrifuge tubes, dried down to completion, and stored at -80°C for polar LC-MS analysis |
| Sampleprep Protocol Filename: | lcms_sequential_extraction_method.pdf |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | IPA for non-polar; 80/20 MeOH/H2O for polar |
| Extract Storage: | -80℃ |
| Sample Resuspension: | 75 µL of IPA containing isotopically labeled lipid standards; 75 µL of 80:20 MeOH: H2O containing isotopically labeled arginine, hypoxanthine, hippuric acid, and methionine |
Combined analysis:
| Analysis ID | AN003416 | AN003417 | AN003418 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | HILIC |
| Chromatography system | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish |
| Column | Thermo Scientific Accucore C30 (150 x 2.1mm,2.6um) | Thermo Scientific Accucore C30 (150 x 2.1mm,2.6um) | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Orbitrap ID-X tribrid | Thermo Orbitrap ID-X tribrid | Thermo Orbitrap ID-X tribrid |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE |
| Units | Ranked Peak Height | Ranked Peak Height | Ranked Peak Height |
Chromatography:
| Chromatography ID: | CH002527 |
| Chromatography Summary: | Non-polar extracts were separated using a Vanquish (ThermoFisher Scientific), fitted with a ThermoFisher Scientific Accucore™ C30 UPLC RP column (2.1 x 150 mm, 2.6 µm particle size). The compounds were eluted with the following gradient: 60:40 acetonitrile:water (ACN:H2O) with 10 mM ammonium formate and 0.1% formic acid (mobile phase A) and 90:10 isopropanol:acetonitrile with 10 mM ammonium formate and 0.1% formic acid (mobile phase B) using the following gradient program: 0.0 min 20% B; 1.0 min 60% B; 5.0 min 70% B; 5.5 min 85% B; 8.0 min 90% B; 8.2-10.5 min 100% B; 10.7-12.0 min 20% B. A curve 5 value was set for 0.0 minutes, and a curve 6 for the remainder of the gradient. The flow rate was set at 0.400 mL min-1. The column temperature was set to 50°C, and the injection volume was 2 µL. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Scientific Accucore C30 (150 x 2.1mm,2.6um) |
| Column Temperature: | 50 |
| Solvent A: | 40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002528 |
| Chromatography Summary: | Polar extracts were separated using a Vanquish (ThermoFisher Scientific), fitted with a Waters Acquity UPLC BEH Amide column (2.1 x 150 mm, 1.7 µm particle size). The compounds were eluted with the following gradient: 80:20 water:acetonitrile (H2O:ACN) with 10 mM ammonium formate and 0.1% formic acid (mobile phase A) and 100% ACN with 0.1% formic acid (mobile phase B) using the following gradient program: 0.0-0.5 min 95% B; 8.0-9.4 min 40% B; 9.5-11.0 min 95% B. A curve 5 value was set for 0.0 minutes, a curve 6 at 0.5 min, curve 7 at 8.0 min, and a curve 6 for the remainder of the gradient. The flow rate was set at 0.400 mL min-1. The column temperature was set to 40°C, and the injection volume was 2 µL. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Column Temperature: | 40 |
| Solvent A: | 80% water/20% acetonitrile; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003181 |
| Analysis ID: | AN003416 |
| Instrument Name: | Thermo Orbitrap ID-X tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | LC-MS data was collected using a Thermo Orbitrap ID-X tribrid in full profile mode at 240,000 resolution. A m/z range of 150-2000 was used. Data was centroided using Proteowizard and processed using open source software SLAW for peak picking, alignment, and feature assignment. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 275 |
| Dry Gas Flow: | 40 |
| Dry Gas Temp: | 320 |
| Spray Voltage: | 3500 |
| Dataformat: | .raw Profile |
| MS ID: | MS003182 |
| Analysis ID: | AN003417 |
| Instrument Name: | Thermo Orbitrap ID-X tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | LC-MS data was collected using a Thermo Orbitrap ID-X tribrid in full profile mode at 240,000 resolution. A m/z range of 150-2000 was used. Data was centroided using Proteowizard and processed using open source software SLAW for peak picking, alignment, and feature assignment. |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 275 |
| Dry Gas Flow: | 40 |
| Dry Gas Temp: | 320 |
| Spray Voltage: | 2500 |
| Dataformat: | .raw Profile |
| MS ID: | MS003183 |
| Analysis ID: | AN003418 |
| Instrument Name: | Thermo Orbitrap ID-X tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | LC-MS data was collected using a Thermo Orbitrap ID-X tribrid in full profile mode at 240,000 resolution. A m/z range of 70-1050 was used. Data was centroided using Proteowizard and processed using open source software SLAW for peak picking, alignment, and feature assignment. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 275 |
| Dry Gas Flow: | 40 |
| Dry Gas Temp: | 320 |
| Spray Voltage: | 3500 |
| Dataformat: | .raw Profile |
