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MB Sample ID: SA076737
Local Sample ID: | BH32_RP.d |
Subject ID: | SU001167 |
Subject Type: | Cells |
Subject Species: | Escherichia coli |
Taxonomy ID: | 562 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001167 |
Subject Type: | Cells |
Subject Species: | Escherichia coli |
Taxonomy ID: | 562 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
BH32_RP.d | SA076737 | FL011734 | Fractionated using RP column | Type of media samples |
Collection:
Collection ID: | CO001161 |
Collection Summary: | Cultures were harvested after 5 days of growth, washed with dH2O, and freeze dried 5 ml of trichloroacetic acid was added to entire freeze dried cell pellet in 15 ml tube, vortexed, and incubated on shaker at 150 rpm for 3 hours TCA was extracted twice w/ 5ml diethyl ether, then sample was washed with 5 ml chloroform, and aqueous phase was recovered Freeze-dried aqueous phase, resuspended in 1 ml MeOH and incubated on shaker for 1 hour at 150 rpm, centrifuged Supernatant was trasnferred to 2 ml tube, sample washed with additional 0.5 ml MeOH and added to 2 ml tube Freeze dried. Note that a method blank and 2 recovery standards containing thiamine were included in this processing |
Collection Protocol Filename: | Thiamine_BH_IG032715QTrap.xlsx |
Sample Type: | Cell |
Treatment:
Treatment ID: | TR001181 |
Treatment Summary: | BH28 was base degraded thiamine. It did not come from an organism. It was suspended thiamine in strong base for an extended period of time. This was done to see what types of degraded products would form. BH29-34 were E. coli medium samples that were fractionated using a reverse phase column. BH35-36 were media samples after E. coli growth where methanol was used for extraction. |
Sample Preparation:
Sampleprep ID: | SP001174 |
Sampleprep Summary: | 1. Add 0.5mL of extraction solvent to tube, gently pipet to remove all cells, transfer cells to 2mL eppendorf tube. Repeat for a total of 1mL extraction solvent + cells in 2mL eppendorf tube. 2. Add 2 small stainless steel grinding beads to eppendorf tube 3. Use the GenoGrinder to grind for 3 minutes at 1,250 rpm. 4. Centrifuge at 14,000xg for 5 minutes. 5. Transfer supernatant to a fresh 2mL eppendorf tube. 6. Add 1mL of extraction solvent to tube containing cell pellet + beads, and repeat steps 3 and 4. 7. Collect supernatant, and combine with supernatant collected in step 5. Total volume of extracted sample will be approximately 2mL. 8. Dry down 50uL of extracted sample in 1.5mL eppendorf tube for GC-TOF analysis. 9. Store backups in -20 or -80C. |
Sampleprep Protocol Filename: | SOP_Extraction_of_Yeast_Cells.pdf |
Combined analysis:
Analysis ID | AN001818 | AN001819 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Agilent 1290 Infinity | Agilent 1290 Infinity |
Column | Waters Acquity BEH C18 (100 x 2.1mm,1.7um) | Waters Acquity BEH C18 (100 x 2.1mm,1.7um) |
MS Type | ESI | ESI |
MS instrument type | QTOF | QTOF |
MS instrument name | Agilent 6530 QTOF | Agilent 6550 QTOF |
Ion Mode | POSITIVE | NEGATIVE |
Units | counts | counts |
Chromatography:
Chromatography ID: | CH001288 |
Instrument Name: | Agilent 1290 Infinity |
Column Name: | Waters Acquity BEH C18 (100 x 2.1mm,1.7um) |
Column Pressure: | 1200 bar |
Column Temperature: | 65 C |
Flow Gradient: | 0% B to 99%B |
Flow Rate: | 0.5 mL/min |
Internal Standard: | CUDA |
Sample Injection: | 3uL |
Solvent A: | 95% water/5% acetonitrile; 0.1% acetic acid |
Solvent B: | 100% acetonitrile; 0.1% acetic acid |
Analytical Time: | 15 min |
Target Sample Temperature: | Autosampler temp 4 C |
Randomization Order: | Excel generated |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS001681 |
Analysis ID: | AN001818 |
Instrument Name: | Agilent 6530 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | none |
Ion Mode: | POSITIVE |
Capillary Voltage: | 3500 |
Collision Gas: | Nitrogen |
Dry Gas Flow: | 8 L/min |
Dry Gas Temp: | 325 C |
Fragment Voltage: | 120 |
Fragmentation Method: | Auto MS/MS |
Ion Source Temperature: | 325 C |
Ion Spray Voltage: | 1000 |
Ionization: | Pos |
Precursor Type: | Intact Molecule |
Reagent Gas: | Nitrogen |
Source Temperature: | 325 C |
Dataformat: | .d |
Desolvation Gas Flow: | 11 L/min |
Desolvation Temperature: | 350 C |
Nebulizer: | 35 psig |
Octpole Voltage: | 750 |
Resolution Setting: | extended dynamic range |
Scan Range Moverz: | 60-1700 Da |
Scanning Cycle: | 2 Hz |
Scanning Range: | 60-1700 Da |
Skimmer Voltage: | 65 |
MS ID: | MS001682 |
Analysis ID: | AN001819 |
Instrument Name: | Agilent 6550 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | none |
Ion Mode: | NEGATIVE |
Capillary Voltage: | 3500 |
Collision Gas: | Nitrogen |
Dry Gas Flow: | 13 L/min |
Dry Gas Temp: | 200 C |
Fragment Voltage: | 175 |
Fragmentation Method: | Auto MS/MS |
Ion Source Temperature: | 325 C |
Ion Spray Voltage: | 1000 |
Ionization: | Neg |
Dataformat: | .d |
Desolvation Gas Flow: | 11 L/min |
Desolvation Temperature: | 350 C |
Nebulizer: | 35 psig |
Octpole Voltage: | 750 |
Scan Range Moverz: | 60-1700 Da |
Scanning Cycle: | 2Hz |
Scanning Range: | 60-1700 |
Skimmer Voltage: | 65 |