Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA250387

Local Sample ID:	1-2
Subject ID:	SU002596
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002596
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
1-2	SA250387	FL031961	1	Time_point(days)

Collection:

Collection ID:	CO002589
Collection Summary:	Murine spleens were harvested and disassociated to single cell suspension at a concentration of 1 x 10^8 cells/mL. Labeling reagent was added at a concentation of 50 μL/mL of sample. Cells were mixed and incubated at RT for 15 mins. Selection cocktail was added to sample at a 70 μL/mL of sample. Cells were mixed and incubated at RT for 15 mins. RapidSpheres were added to sample at a 50 μL/mL of sample. Cells were mixed and incubated at RT for 10 mins. Cells were washed with FACS buffer for 4 times, and were resuspended for downstream metabolomics analysis.
Sample Type:	Stem cells

Treatment:

Treatment ID:	TR002608
Treatment Summary:	Phenylhydrazine (PHZ)-induced hemolytic anemia To induce acute hemolytic anemia, age- and sex-matched mice were injected intraperitoneally with a single dose (100 mg/kg body weight) of freshly prepared phenylhydrazine (Sigma-Aldrich, dissolved in PBS). Mice were sacrificed at indicated time points for spleen sample collection and Kit cell enrichment. Brucella abortus-induced inflammatory anemia Heat-killed Brucella abortus (HKBA, strain 1119-3) was used to induce anemia of inflammation following a previously described method(1). To induce stress erythropoiesis, age- and sex-matched mice were administered with 5 x 108 particles of HKBA via intraperitoneal injection.Mice were sacrificed at indicated time points for spleen sample collection and Kit cell enrichment.

Treatment ID:

TR002608

Treatment Summary:

Phenylhydrazine (PHZ)-induced hemolytic anemia To induce acute hemolytic anemia, age- and sex-matched mice were injected intraperitoneally with a single dose (100 mg/kg body weight) of freshly prepared phenylhydrazine (Sigma-Aldrich, dissolved in PBS). Mice were sacrificed at indicated time points for spleen sample collection and Kit cell enrichment. Brucella abortus-induced inflammatory anemia Heat-killed Brucella abortus (HKBA, strain 1119-3) was used to induce anemia of inflammation following a previously described method(1). To induce stress erythropoiesis, age- and sex-matched mice were administered with 5 x 108 particles of HKBA via intraperitoneal injection.Mice were sacrificed at indicated time points for spleen sample collection and Kit cell enrichment.

Sample Preparation:

Sampleprep ID:	SP002602
Sampleprep Summary:	Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample.

Sampleprep ID:

SP002602

Sampleprep Summary:

Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample.

Combined analysis:

Analysis ID	AN004106
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000
Column	Waters XBridge C18 (50 x 2.1mm,3um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap
Ion Mode	NEGATIVE
Units	peak area normalized to IS and cell number

Chromatography:

Chromatography ID:	CH003041
Chromatography Summary:	The sample run order was randomized to reduce bias from instrument drift. 10 µl sample was subjected to LC-MS analysis on a Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Reversed-phase chromatography mode was used to separate compounds on a Xselect C18 HSS column (Waters) with solvent A (97:3 water:methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid ) and solvent B (methanol). The flow rate was 200 µl/min, and the total run time was 25 min. The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B. The mass spectrometer was operated in a negative-ion mode at a resolution of 140,000 at m/z 200 and with a scan range of 85 to 1000 m/z.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters XBridge C18 (50 x 2.1mm,3um)
Column Temperature:	standard
Flow Gradient:	0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B.
Flow Rate:	200 µl/min
Solvent A:	97:3 water:methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid
Solvent B:	methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003853
Analysis ID:	AN004106
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data files were converted to .mzML file format using the ProteoWizard software, and they were analyzed by the MS-DIAL software. Metabolites were identified by comparison to an in-house reference library of pure metabolite standards which included mass-to-charge ratio (m/z) and retention time. For quantification of metabolite abundance, peak areas of identified metabolites were first normalized to the internal standard chlorpropamide, and then normalized to cell numbers from each sample. Data were analyzed using R and Cytoscape software.
Ion Mode:	NEGATIVE