Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA210574

Local Sample ID:	5
Subject ID:	SU002283
Subject Type:	Mammal
Subject Species:	Mus musculus

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Subject:

Subject ID:	SU002283
Subject Type:	Mammal
Subject Species:	Mus musculus

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
5	SA210574	FL025718	CIM	Factor

Collection:

Collection ID:	CO002276
Collection Summary:	C57BL/6 mice were purchased from OrientBio (Sungnam, Korea). Female mice, ages 8 to 10 weeks, were immunized intradermally with 200 μg of the C-protein fragments emulsified in complete Freund’s adjuvant (CFA) containing 100 μg of heat-killed Mycobacterium butyricum (Difco, Franklin Lakes, NJ, USA). Mice were sacrificed and proximal muscles (hamstring and quadriceps) of both hind legs were harvested.
Sample Type:	muscle tissue

Treatment:

Treatment ID:	TR002295
Treatment Summary:	N/A

Sample Preparation:

Sampleprep ID:	SP002289
Sampleprep Summary:	Centrifuge the calibration standards, QC, and ISTD mix vials before opening at 10 000 x g (rcf) for 2 min. Dissolve calibration standards (red caps) in 100 µL water. Dissolve QCs (green, blue and yellow caps) in 100 µL water. Dissolve ISTD mix (orange cap) in 1200 µL water. Shake all vials (Cal, QCs, ISTD) for 15 min at 1200 rpm and vortex several times. Centrifuge the QCs and your plasma samples for 5 min at 2750 x g (rcf) and 4 °C. Add 10 µL of the ISTD mix to each well of the Kit plate except A1. Pipette 10 µL PBS / Cal / QC / sample according to your plate layout generated by MetIDQTM. Dry for 30 min under nitrogen (using a Nitrogen Evaporator or a Pressure Manifold). Prepare the pre-mix for derivatization in the plastic tube of the Kit box (1900 µL of each: ethanol, water and pyridine). Add 300 µL phenylisothiocyanate (PITC) and vortex rigorously (at least 10 sec) until the derivatization solution is clear. Make half of the solution for the starter kit. Add 50 µL of the derivatization solution to each well using a repeater at maximum dispensing speed. Cover the plate with the plastic lid and incubate for 25 min, then dry for 60 min under nitrogen. Prepare extraction solvent and add 300 µL to each well. You can use the repeater at low dispensing speed or an 8-channel pipette. Shake for 30 min at 450 rpm (on the scale of min-max 0-1400 rpm). Centrifuge for 2 min at 500 x g (rcf) or use a Pressure Manifold. Visually check that the fill level is the same in all wells of the capture plate. If not, repeat and apply higher g or higher pressure, respectively. Carefully remove the upper filter plate and transfer the volume specified for your instrument to another empty 96-deep well plate that you find in the Kit box (label “LC plate”). Use an 8-channel pipette. Finish the LC plate and dilute the extracts as described for your instrument Prepare the FIA plate and dilute the original extracts as described for your instrument Seal both plates with the silicon mat. Firmly press the mat’s naps into the wells. Shake both plates for 2 min at 600 rpm. Put the plates in the autosampler.

Sampleprep ID:

SP002289

Sampleprep Summary:

Centrifuge the calibration standards, QC, and ISTD mix vials before opening at 10 000 x g (rcf) for 2 min. Dissolve calibration standards (red caps) in 100 µL water. Dissolve QCs (green, blue and yellow caps) in 100 µL water. Dissolve ISTD mix (orange cap) in 1200 µL water. Shake all vials (Cal, QCs, ISTD) for 15 min at 1200 rpm and vortex several times. Centrifuge the QCs and your plasma samples for 5 min at 2750 x g (rcf) and 4 °C. Add 10 µL of the ISTD mix to each well of the Kit plate except A1. Pipette 10 µL PBS / Cal / QC / sample according to your plate layout generated by MetIDQTM. Dry for 30 min under nitrogen (using a Nitrogen Evaporator or a Pressure Manifold). Prepare the pre-mix for derivatization in the plastic tube of the Kit box (1900 µL of each: ethanol, water and pyridine). Add 300 µL phenylisothiocyanate (PITC) and vortex rigorously (at least 10 sec) until the derivatization solution is clear. Make half of the solution for the starter kit. Add 50 µL of the derivatization solution to each well using a repeater at maximum dispensing speed. Cover the plate with the plastic lid and incubate for 25 min, then dry for 60 min under nitrogen. Prepare extraction solvent and add 300 µL to each well. You can use the repeater at low dispensing speed or an 8-channel pipette. Shake for 30 min at 450 rpm (on the scale of min-max 0-1400 rpm). Centrifuge for 2 min at 500 x g (rcf) or use a Pressure Manifold. Visually check that the fill level is the same in all wells of the capture plate. If not, repeat and apply higher g or higher pressure, respectively. Carefully remove the upper filter plate and transfer the volume specified for your instrument to another empty 96-deep well plate that you find in the Kit box (label “LC plate”). Use an 8-channel pipette. Finish the LC plate and dilute the extracts as described for your instrument Prepare the FIA plate and dilute the original extracts as described for your instrument Seal both plates with the silicon mat. Firmly press the mat’s naps into the wells. Shake both plates for 2 min at 600 rpm. Put the plates in the autosampler.

Combined analysis:

Analysis ID	AN003596
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	AB SCIEX QTRAP 5500
Column	Waters ACQUITY UPLC BEH C18 (75 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	QTRAP
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	POSITIVE
Units	ng/mL

Chromatography:

Chromatography ID:	CH002657
Instrument Name:	AB SCIEX QTRAP 5500
Column Name:	Waters ACQUITY UPLC BEH C18 (75 x 2.1mm,1.7um)
Solvent A:	100% water; 0.2% formic acid
Solvent B:	100% acetonitrile; 0.2% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003351
Analysis ID:	AN003596
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	For LC part: Mobile phase A: 1000 mL water + 2 mL formic acid (FA) Mobile phase B: 500 mL ACN + 1 mL FA Wash solvent 1: 500 mL ACN + 200 mL MeOH + 150 mL iPrOH + 150 mL water + 2.5 mL FA Wash solvent 2: water Install column and precolumn (if required). Wash the column at 95% B and condition to 100% For FIA part: Deactivate “Use flat files for scan data” in the Analyst software FIA mobile phase: FIA Mobile Phase Additive (1x glass ampule) + 290 mL MeOH Wash solvent 1: 500 mL ACN + 200 mL MeOH + 150 mL iPrOH + 150 mL water + 2.5 mL FA Wash solvent 2 (if possible): 333 mL MeOH + 333 mL iPrOH + 333 mL water For data processing: Adjust the retention times in the quantitation method and if necessary some integration parameters. Process the LC-MS data using the Quantitation Wizard. Save the results. Import the results export (TXT file) into MetIDQTM  Parse the FIA-MS WIFF files directly into MetIDQTM. Evaluate results, perform statistics (e.g. using MetaboINDICATOR™, StatPack, MetaboAnalyst).
Ion Mode:	POSITIVE