Summary of Study ST004306

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002722. The data can be accessed directly via it's Project DOI: 10.21228/M8T54K This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study ID	ST004306
Study Title	Comparative Metabolomic Profiling of Bone Marrow-Derived Macrophages from Wild-Type and MG53-KO Mice
Study Summary	MG53 whole-body knockout (KO) mice and their wild-type (WT) littermates were bred and housed under specific pathogen-free conditions. Bone marrow-derived macrophages (BMDMs) were isolated from four biologically independent 8-week-old mice per genotype (N=4). Following isolation, cells were plated with bone marrow culture medium (10% FBS in DMEM, 1% penicillin streptomycin, 50ng ml-1 M-CSF (Abclonal, #RP01216)). Cells were allowed to attach to culture dishes within 48 hours. The culture medium was refreshed on day 3 to support differentiation and growth. The total experimental duration in vitro was 5 days. On day 5, the fully differentiated BMDMs were harvested by trypsinization. The resulting cell pellets were collected, snap-frozen, and stored at -80°C to preserve metabolic integrity for subsequent metabolomic analysis. This well-controlled study design, with biological replication (N=4), ensured the reliable identification of metabolomic alterations directly attributable to MG53 deficiency in macrophages.
Institute	Zhejiang University
Last Name	Pei
First Name	Yumeng
Address	Wu Tong Road Number 366, HangZhou, Zhejiang, China, 310027
Email	0623B01@zju.edu.cn
Phone	(0571)87236114
Submit Date	2025-10-22
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-10-28
Release Version	1

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Project:

Project ID:	PR002722
Project DOI:	doi: 10.21228/M8T54K
Project Title:	Comparative Metabolomic Profiling of Bone Marrow-Derived Macrophages from Wild-Type and MG53-KO Mice
Project Summary:	Tumor-associated macrophages (TAMs) are pivotal players in the tumor microenvironment. Our preliminary research has identified the expression of MG53 (Mitsugumin 53) in TAMs. Through a series of in vitro and in vivo experiments, we have initially demonstrated that the genetic ablation of MG53 drives macrophages toward a pro-tumoric M2-like phenotypic polarization. The primary objective of this project was to investigate the underlying metabolic reprogramming associated with MG53 deficiency in macrophages. We hypothesized that the MG53-mediated M2 polarization would be underpinned by a distinct metabolic profile. This study aimed to comprehensively characterize the metabolomic landscape of bone marrow-derived macrophages (BMDMs) from MG53-knockout (KO) mice compared to their wild-type (WT) littermates to validate this hypothesis at the metabolic level. Our metabolomic analysis successfully revealed a definitive M2-like metabolic signature in MG53-KO BMDMs. This provides crucial functional validation of our prior findings and establishes a direct link between MG53 and metabolic reprogramming in macrophage polarization. These results position MG53 as a novel and critical regulator at the nexus of immunometabolism, suggesting its potential as a therapeutic target for modulating macrophage function in cancer.
Institute:	Zhejiang University
Last Name:	Pei
First Name:	Yumeng
Address:	Wu Tong Road Number 366, HangZhou, Zhejiang, China, 310027
Email:	0623B01@zju.edu.cn
Phone:	(0571)87236114

Subject:

Subject ID:	SU004460
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype	Sample source
SA505358	KO_1	MG53-knockout	Bone marrow derived macrophages
SA505359	KO_2	MG54-knockout	Bone marrow derived macrophages
SA505360	KO_3	MG55-knockout	Bone marrow derived macrophages
SA505361	KO_4	MG56-knockout	Bone marrow derived macrophages
SA505362	WT_1	Wild-type	Bone marrow derived macrophages
SA505363	WT_2	Wild-type	Bone marrow derived macrophages
SA505364	WT_3	Wild-type	Bone marrow derived macrophages
SA505365	WT_4	Wild-type	Bone marrow derived macrophages

Showing results 1 to 8 of 8

Showing results 1 to 8 of 8

Collection:

Collection ID:	CO004453
Collection Summary:	Bone marrow-derived macrophages (BMDMs) were isolated from MG53-knockout (KO) mice and their wild-type (WT) littermates. After 5 days of in vitro culture, the culture supernatant was removed. Adherent BMDMs were then harvested by trypsinization and collected for subsequent metabolomic analysis.
Sample Type:	Macrophages

Treatment:

Treatment ID:	TR004469
Treatment Summary:	Bone marrow-derived macrophages (BMDMs) were isolated from MG53-knockout (KO) mice and their wild-type (WT) littermates. Cells were cultured in vitro for 5 days to allow for differentiation and stabilization, after which they were harvested for metabolomic analysis.

Sample Preparation:

Sampleprep ID:	SP004466
Sampleprep Summary:	Methanol (0.75 mL) was added to a 100 μL sample , which was placed into a glass tube with a Teflon lined cap, and the tube was vortexed. 2.5 mL of MTBE was added, then 10 μL of SPLASH™ internal standard was added and the mixture was incubated for 1 h at room temperature in a shaker. Phase separation was induced by adding 0.625 mL of MS-grade water. Upon 10 min of incubation at room temperature, the sample was centrifuged at 1,000 g for 10 min. The upper (organic) phase was collected, and the lower phase was re-extracted with 1 mL of the solvent mixture（MTBE/methanol/water (10:3:2.5, v/v/v)), and collecting the upper phase. Combined organicphases were dried and dissolved in 100 μL of isopropanol for storage. Then analyzed by LC-MS/MS. UHPLC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher, Germany) coupled with an Orbitrap Q ExactiveTM HF mass spectrometer (Thermo Fisher, Germany). Samples were injected onto a Thermo Accucore C30 column (150 x 2.1 mm, 2.6 μm) using a 20-min linear gradient at a flow rate of 0.35 mL/min. The column temperature was set at 40℃. Mobile phase buffer A was acetonitrile / water(6/4) with 10 mM ammonium acetate and 0.1% formic acid, whereas buffer B was acetonitrile/isopropanol (1/9) with 10 mM ammonium acetate and 0.1% formic acid. The solvent gradient was set as follows: 30% B, initial; 30% B, 2min; 43% B, 5 min；55% B, 5.1 min；70% B, 11 min；99%B, 16 min;30%B, 18.1min. Q Exactive HF mass spectrometer was operated in positive [negative] polarity mode with sheath gas :40 psi, sweep gas: 0 L/min, auxiliary gasrate: 10 L/min [7 L/min], spray voltage: 3.5 kV, capillary temperature: 320℃, heater temperature: 350℃, S-Lens RF level: 50, scan range: 114–1700 m/z, automatic gain control target: 3e6, normalized collision energy: 22eV; 24 eV; 28eV [22 eV；24 eV；28 eV], Injection time: 100 ms, Isolation window: 1m/z, automatic gaincontrol target（MS2）: 2e5, dynamic exclusion: 6s.

Sampleprep ID:

SP004466

Sampleprep Summary:

Methanol (0.75 mL) was added to a 100 μL sample , which was placed into a glass tube with a Teflon lined cap, and the tube was vortexed. 2.5 mL of MTBE was added, then 10 μL of SPLASH™ internal standard was added and the mixture was incubated for 1 h at room temperature in a shaker. Phase separation was induced by adding 0.625 mL of MS-grade water. Upon 10 min of incubation at room temperature, the sample was centrifuged at 1,000 g for 10 min. The upper (organic) phase was collected, and the lower phase was re-extracted with 1 mL of the solvent mixture（MTBE/methanol/water (10:3:2.5, v/v/v)), and collecting the upper phase. Combined organicphases were dried and dissolved in 100 μL of isopropanol for storage. Then analyzed by LC-MS/MS. UHPLC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher, Germany) coupled with an Orbitrap Q ExactiveTM HF mass spectrometer (Thermo Fisher, Germany). Samples were injected onto a Thermo Accucore C30 column (150 x 2.1 mm, 2.6 μm) using a 20-min linear gradient at a flow rate of 0.35 mL/min. The column temperature was set at 40℃. Mobile phase buffer A was acetonitrile / water(6/4) with 10 mM ammonium acetate and 0.1% formic acid, whereas buffer B was acetonitrile/isopropanol (1/9) with 10 mM ammonium acetate and 0.1% formic acid. The solvent gradient was set as follows: 30% B, initial; 30% B, 2min; 43% B, 5 min；55% B, 5.1 min；70% B, 11 min；99%B, 16 min;30%B, 18.1min. Q Exactive HF mass spectrometer was operated in positive [negative] polarity mode with sheath gas :40 psi, sweep gas: 0 L/min, auxiliary gasrate: 10 L/min [7 L/min], spray voltage: 3.5 kV, capillary temperature: 320℃, heater temperature: 350℃, S-Lens RF level: 50, scan range: 114–1700 m/z, automatic gain control target: 3e6, normalized collision energy: 22eV; 24 eV; 28eV [22 eV；24 eV；28 eV], Injection time: 100 ms, Isolation window: 1m/z, automatic gaincontrol target（MS2）: 2e5, dynamic exclusion: 6s.

Combined analysis:

Analysis ID	AN007165	AN007166
Chromatography ID	CH005443	CH005443
MS ID	MS006860	MS006861
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish UHPLC	Thermo Vanquish UHPLC
Column	Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm)	Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF-X Orbitrap	Thermo Q Exactive HF-X Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	μg/g	μg/g

Chromatography:

Chromatography ID:	CH005443
Instrument Name:	Thermo Vanquish UHPLC
Column Name:	Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm)
Column Temperature:	40℃
Flow Gradient:	30% B, initial; 30% B, 2min; 43% B, 5 min；55% B, 5.1 min；70% B, 11 min； 99%B, 16 min;30%B, 18.1min.
Flow Rate:	0.35 mL/min
Solvent A:	60% Acetonitrile/40% Water; 10 mM ammonium acetate; 0.1% formic acid
Solvent B:	10% Acetonitrile/90% Isopropanol; 10 mM ammonium acetate; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS006860
Analysis ID:	AN007165
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Q ExactiveTM HF mass spectrometer was operated in positive [negative] polarity mode with sheath gas :40 psi, sweep gas: 0 L/min, auxiliary gasrate: 10 L/min [7 L/min], spray voltage: 3.5 kV, capillary temperature: 320℃, heater temperature: 350℃, S-Lens RF level: 50, scan range: 114–1700 m/z, automatic gain control target: 3e6, normalized collision energy: 22eV; 24 eV; 28eV [22 eV；24 eV；28 eV], Injection time: 100 ms, Isolation window: 1m/z, automatic gaincontrol target（MS2）: 2e5, dynamic exclusion: 6s.
Ion Mode:	POSITIVE

MS ID:	MS006861
Analysis ID:	AN007166
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Q ExactiveTM HF mass spectrometer was operated in positive [negative] polarity mode with sheath gas :40 psi, sweep gas: 0 L/min, auxiliary gasrate: 10 L/min [7 L/min], spray voltage: 3.5 kV, capillary temperature: 320℃, heater temperature: 350℃, S-Lens RF level: 50, scan range: 114–1700 m/z, automatic gain control target: 3e6, normalized collision energy: 22eV; 24 eV; 28eV [22 eV；24 eV；28 eV], Injection time: 100 ms, Isolation window: 1m/z, automatic gaincontrol target（MS2）: 2e5, dynamic exclusion: 6s.
Ion Mode:	NEGATIVE