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MB Sample ID: SA194018

Local Sample ID:	KR2M2
Subject ID:	SU002143
Subject Type:	Cultured cells
Subject Species:	Mus musculus

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Subject:

Subject ID:	SU002143
Subject Type:	Cultured cells
Subject Species:	Mus musculus

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
KR2M2	SA194018	FL023767	KR	Genotype

Collection:

Collection ID:	CO002136
Collection Summary:	The labelled cells were collected and washed with 2 mL of PBS, and then were quenched with 2 mL methanol (-20℃). To increase the polarity for the two phase extraction, 0.4 mL of 4℃ cold water was added.
Sample Type:	Macrophages

Treatment:

Treatment ID:	TR002155
Treatment Summary:	Method was described in previous studies (Jha et al., 2015; Lauterbach et al., 2019). 107 BMDMs per group were seeded in 10cm plates and incubated in RPMI-1640 cell culture medium with 10% FBS. Prior to isotopic labeling, the medium was replaced with RPMI-1640 without glutamine for 4 hrs. Then stable-isotope labeled analog 4 mM (U-13C5) glutamine (Cambridge Isotope) was added together with IL-4 (20ng/ml) for 4 hrs.

Treatment ID:

TR002155

Treatment Summary:

Method was described in previous studies (Jha et al., 2015; Lauterbach et al., 2019). 107 BMDMs per group were seeded in 10cm plates and incubated in RPMI-1640 cell culture medium with 10% FBS. Prior to isotopic labeling, the medium was replaced with RPMI-1640 without glutamine for 4 hrs. Then stable-isotope labeled analog 4 mM (U-13C5) glutamine (Cambridge Isotope) was added together with IL-4 (20ng/ml) for 4 hrs.

Sample Preparation:

Sampleprep ID:	SP002149
Sampleprep Summary:	The samples were processed by 5 cycles of 1 min ultra-sonication and 1 min interval in ice-water bath. After that samples were stood for 30 min at -40℃ and 10 min at 4 ℃. After centrifugation at 15000 g and 4℃ for 15 min. The supernatant was evaporated to dryness under mild nitrogen and reconstituted in 50 L of 50% acetonitrile (including 1g/mL phenylalanine-d5 internal standard) prior to perform further analysis.

Sampleprep ID:

SP002149

Sampleprep Summary:

The samples were processed by 5 cycles of 1 min ultra-sonication and 1 min interval in ice-water bath. After that samples were stood for 30 min at -40℃ and 10 min at 4 ℃. After centrifugation at 15000 g and 4℃ for 15 min. The supernatant was evaporated to dryness under mild nitrogen and reconstituted in 50 L of 50% acetonitrile (including 1g/mL phenylalanine-d5 internal standard) prior to perform further analysis.

Combined analysis:

Analysis ID	AN003360
Analysis type	MS
Chromatography type	HILIC
Chromatography system	ThermoFisher Ultimate 3000 UHPLC system
Column	Waters BEH Amide
MS Type	ESI
MS instrument type	Hybrid Quadrupole-Orbitrap™
MS instrument name	ThermoFisher Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometry (QE)
Ion Mode	NEGATIVE
Units	pmoles/I

Chromatography:

Chromatography ID:	CH002486
Chromatography Summary:	Chromatographic separation was performed on a ThermoFisher Ultimate 3000 UHPLC system with a Waters BEH Amide column (2.1mm × 100 mm, 1.7 μm). The injection volume was 2μL and the flow rate was 0.25 mL/min. The column temperature was 15°C. The mobile phases consisted of water with 0.01% formic acid and 2 mM ammonium formate (phase A) and acetonitrile (phase B). A linear gradient elution was performed with the following program: 0 min, 90%B; 4 min, 85% B; 11 min, 75%B; 14 min, 70%B, 14.5min, 50%B and held to 17 min; 17.1 min, 90%B and held to 20.01 min.
Instrument Name:	ThermoFisher Ultimate 3000 UHPLC system
Column Name:	Waters BEH Amide
Column Temperature:	15
Flow Gradient:	0 min, 90%B; 4 min, 85% B; 11 min, 75%B; 14 min, 70%B, 14.5min, 50%B and held to 17 min; 17.1 min, 90%B and held to 20.01 min.
Flow Rate:	0.25ml/min
Solvent A:	100% water; 0.01% formic acid; 2 mM ammonium formate
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS003129
Analysis ID:	AN003360
Instrument Name:	ThermoFisher Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometry (QE)
Instrument Type:	Hybrid Quadrupole-Orbitrap™
MS Type:	ESI
MS Comments:	The eluents were analyzed on a ThermoFisher Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometry (QE) in Heated Electrospray Ionization Negative (HESI-) mode, separately. Spray voltage was set to 4000 V. Capillary and Probe Heater Temperature were separately 320 °C and 320 °C. Sheath gas flow rate was 35 (Arb, arbitrary unit), and Aux gas flow rate was 10 (Arb). S-Lens RF Level was 50 (Arb). The full scan was operated at a high-resolution of 70000 FWHM (m/z=200) at a range of 70- 1050 m/z with AGC Target setting at 3×106.
Ion Mode:	NEGATIVE