Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA171159

Local Sample ID:	AW_F_CO_6h_014
Subject ID:	SU001917
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Not applicable

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Subject:

Subject ID:	SU001917
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_F_CO_6h_014	SA171159	FL020612	Control	treatment

Collection:

Collection ID:	CO001910
Collection Summary:	animals sacrificed 2, 6, and 24 hours following naphthalene treatment.
Sample Type:	Liver

Treatment:

Treatment ID:	TR001929
Treatment Summary:	Subjects were divided by 2, 6, and 24 hours following naphthalene and control

Sample Preparation:

Sampleprep ID:	SP001923
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.

Sampleprep ID:

SP001923

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	AN002983
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 6550
Column	Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type	EI
MS instrument type	CSH
MS instrument name	Agilent 6550 QTOF
Ion Mode	UNSPECIFIED
Units	normalized peak height

Chromatography:

Chromatography ID:	CH002212
Chromatography Summary:	Complex lipids by LC QTOF CSH
Instrument Name:	Agilent 6550
Column Name:	Waters Acquity BEH C18 (100 x 2mm,1.7um)
Column Temperature:	65
Flow Gradient:	0 min 85% (A); 0-2 min 70% (A); 2-2.5 min 52% (A); 2.5-11 min 18% (A); 11-11.5 min 1% (A); 11.5-12 min 1% (A); 12-12.1 min 85% (A); 12.1-15 min 85% (A)
Flow Rate:	0.6 mL/min
Solvent A:	60% acetonitrile/40% water; 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

MS:

MS ID:	MS002773
Analysis ID:	AN002983
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	CSH
MS Type:	EI
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) coupled to either an Agilent 6530 (positive ion mode) or an Agilent 6550 mass spectrometer equipped with an ion funnel (iFunnel) (negative ion mode). Lipids are separated on an Acquity UPLC CSH C18 column (100 x 2.1 mm; 1.7 µm) maintained at 65°C at a flow-rate of 0.6 mL/min. Solvent pre-heating (Agilent G1316) was used. The mobile phases consist of 60:40 acetonitrile:water with 10 mM ammonium formate and 0.1% formic acid (A) and 90:10 propan-2-ol:acetonitrile with 10 mM ammonium formate and 0.1% formic acid. The gradient is as follows: 0 min 85% (A); 0–2 min 70% (A); 2–2.5 min 52% (A); 2.5–11 min 18% (A); 11–11.5 min 1% (A); 11.5–12 min 1% (A); 12–12.1 min 85% (A); 12.1–15 min 85% (A). A sample volume of 3 µL is used for the injection. Sample temperature is maintained at 4°C in the autosampler. The quadrupole/time-of-flight (QTOF) mass spectrometers are operated with electrospray ionization (ESI) performing full scan in the mass range m/z 65–1700 in positive (Agilent 6530, equipped with a JetStreamSource) and negative (Agilent 6550, equipped with a dual JetStream Source) modes producing both unique and complementary spectra. Instrument parameters are as follows (positive mode) Gas Temp 325°C, Gas Flow 8 l/min, Nebulizer 35 psig, Sheath Gas 350°C, Sheath Gas Flow 11, Capillary Voltage 3500 V, Nozzle Voltage 1000V, Fragmentor 120V, Skimmer 65V. Data (both profile and centroid) are collected at a rate of 2 scans per second. In negative ion mode, Gas Temp 200°C, Gas Flow 14 l/min, Fragmentor 175V, with the other parameters identical to positive ion mode. For the 6530 QTOF, a reference solution generating ions of 121.050 and 922.007 m/z in positive mode and 119.036 and 966.0007 m/z in negative mode, and these are used for continuous mass correction. For the 6550, the reference solution is introduced via a dual spray ESI, with the same ions and continuous mass correction. Samples are injected (1.7 μl in positive mode and 5 μl in negative ion mode) with a needle wash for 20 seconds (wash solvent is isopropanol). The valve is switched back and forth during the run for washing; this has been shown to be essential for reducing carryover of less polar lipids.
Ion Mode:	UNSPECIFIED