Return to study ST001841 main page
MB Sample ID: SA171329
Local Sample ID: | AW_M_CO_24h_040 |
Subject ID: | SU001918 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Not applicable |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001918 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Not applicable |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
AW_M_CO_24h_040 | SA171329 | FL020614 | Control | treatment |
Collection:
Collection ID: | CO001911 |
Collection Summary: | animals sacrificed 2, 6, and 24 hours following naphthalene treatment. |
Sample Type: | Liver |
Treatment:
Treatment ID: | TR001930 |
Treatment Summary: | Subjects were divided by 2, 6, and 24 hours following naphthalene and control |
Sample Preparation:
Sampleprep ID: | SP001924 |
Sampleprep Summary: | Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules. |
Combined analysis:
Analysis ID | AN002984 |
---|---|
Analysis type | MS |
Chromatography type | HILIC |
Chromatography system | Agilent 6550 |
Column | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
MS Type | ESI |
MS instrument type | QTOF |
MS instrument name | Agilent 6550 QTOF |
Ion Mode | UNSPECIFIED |
Units | normalized peak height |
Chromatography:
Chromatography ID: | CH002213 |
Chromatography Summary: | Biogenic amines by LC QTOF HILIC |
Instrument Name: | Agilent 6550 |
Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
Column Temperature: | 45 |
Flow Gradient: | 0 min 100% (A); 0-2 min 100% (A); 2-7.7 min 30% (A); 7.7-9.5 min 60% (A); 9.5-10.3 min 70% (A); 10.3-12.8 min 0% (A); 12.8-16.8 min 0% (A) |
Flow Rate: | 0.4 mL/min |
Solvent A: | 100% water; 0.125% formic acid; 10 mM ammonium formate |
Solvent B: | 95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate |
Chromatography Type: | HILIC |
MS:
MS ID: | MS002774 |
Analysis ID: | AN002984 |
Instrument Name: | Agilent 6550 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | LC/MS parameters The LC/QTOFMS analyses are performed using an Agilent 1290 Infinity LC system (G4220A binary pump, G4226A autosampler, and G1316C Column Thermostat). Polar compounds are separated on an Acquity UPLC BEH Amide Column, 130Å, 1.7 µm, 2.1 mm X 150 mm maintained at 45°C at a flow-rate of 0.4 mL/min. Solvent pre-heating (Agilent G1316) was used. The mobile phases consist of: Water, 10 mM Ammonium Formate, 0.125% Formic Acid (A) and Acetonitrile: Water (95/5, v/v), 10 mM Ammonium Formate, 0.125% Formic Acid (B) The gradient is as follows: 0 min 100% (A); 0–2 min 100% (A); 2–7.7 min 30% (A); 7.7–9.5 min 60% (A); 9.5–10.3 min 70% (A); 10.3–12.8 min 0% (A); 12.8–16.8 min 0% (A. A sample volume of 1 µL for positive mode and 3 µL for negative mode is used for the injection. Sample temperature is maintained at 4°C in the autosampler. spectrometers are operated with electrospray ionization (ESI) performing full scan in the mass range m/z 50–1200. Number of cycles in MS1 is 1667 with cycle time of 500ms and accumulation time 475ms. Mass spectrometer parameters are as follows (positive mode) Gas Temp 300°C, gas pressures in psi units with : GS1 and GS2 50 psi, CUR: 35. ISVF is 4500V and DP and CE are 10V and 100 V. |
Ion Mode: | UNSPECIFIED |