#METABOLOMICS WORKBENCH YumengPei_20251022_060123 DATATRACK_ID:6586 STUDY_ID:ST004306 ANALYSIS_ID:AN007166 PROJECT_ID:PR002722 VERSION 1 CREATED_ON October 22, 2025, 6:50 pm #PROJECT PR:PROJECT_TITLE Comparative Metabolomic Profiling of Bone Marrow-Derived Macrophages from PR:PROJECT_TITLE Wild-Type and MG53-KO Mice PR:PROJECT_SUMMARY Tumor-associated macrophages (TAMs) are pivotal players in the tumor PR:PROJECT_SUMMARY microenvironment. Our preliminary research has identified the expression of MG53 PR:PROJECT_SUMMARY (Mitsugumin 53) in TAMs. Through a series of in vitro and in vivo experiments, PR:PROJECT_SUMMARY we have initially demonstrated that the genetic ablation of MG53 drives PR:PROJECT_SUMMARY macrophages toward a pro-tumoric M2-like phenotypic polarization. The primary PR:PROJECT_SUMMARY objective of this project was to investigate the underlying metabolic PR:PROJECT_SUMMARY reprogramming associated with MG53 deficiency in macrophages. We hypothesized PR:PROJECT_SUMMARY that the MG53-mediated M2 polarization would be underpinned by a distinct PR:PROJECT_SUMMARY metabolic profile. This study aimed to comprehensively characterize the PR:PROJECT_SUMMARY metabolomic landscape of bone marrow-derived macrophages (BMDMs) from PR:PROJECT_SUMMARY MG53-knockout (KO) mice compared to their wild-type (WT) littermates to validate PR:PROJECT_SUMMARY this hypothesis at the metabolic level. Our metabolomic analysis successfully PR:PROJECT_SUMMARY revealed a definitive M2-like metabolic signature in MG53-KO BMDMs. This PR:PROJECT_SUMMARY provides crucial functional validation of our prior findings and establishes a PR:PROJECT_SUMMARY direct link between MG53 and metabolic reprogramming in macrophage polarization. PR:PROJECT_SUMMARY These results position MG53 as a novel and critical regulator at the nexus of PR:PROJECT_SUMMARY immunometabolism, suggesting its potential as a therapeutic target for PR:PROJECT_SUMMARY modulating macrophage function in cancer. PR:INSTITUTE Zhejiang University PR:LAST_NAME Pei PR:FIRST_NAME Yumeng PR:ADDRESS Wu Tong Road Number 366 PR:EMAIL 0623B01@zju.edu.cn PR:PHONE (0571)87236114 #STUDY ST:STUDY_TITLE Comparative Metabolomic Profiling of Bone Marrow-Derived Macrophages from ST:STUDY_TITLE Wild-Type and MG53-KO Mice ST:STUDY_SUMMARY MG53 whole-body knockout (KO) mice and their wild-type (WT) littermates were ST:STUDY_SUMMARY bred and housed under specific pathogen-free conditions. Bone marrow-derived ST:STUDY_SUMMARY macrophages (BMDMs) were isolated from four biologically independent 8-week-old ST:STUDY_SUMMARY mice per genotype (N=4). Following isolation, cells were plated with bone marrow ST:STUDY_SUMMARY culture medium (10% FBS in DMEM, 1% penicillin streptomycin, 50ng ml-1 M-CSF ST:STUDY_SUMMARY (Abclonal, #RP01216)). Cells were allowed to attach to culture dishes within 48 ST:STUDY_SUMMARY hours. The culture medium was refreshed on day 3 to support differentiation and ST:STUDY_SUMMARY growth. The total experimental duration in vitro was 5 days. On day 5, the fully ST:STUDY_SUMMARY differentiated BMDMs were harvested by trypsinization. The resulting cell ST:STUDY_SUMMARY pellets were collected, snap-frozen, and stored at -80°C to preserve metabolic ST:STUDY_SUMMARY integrity for subsequent metabolomic analysis. This well-controlled study ST:STUDY_SUMMARY design, with biological replication (N=4), ensured the reliable identification ST:STUDY_SUMMARY of metabolomic alterations directly attributable to MG53 deficiency in ST:STUDY_SUMMARY macrophages. ST:INSTITUTE Zhejiang University ST:LAST_NAME Pei ST:FIRST_NAME Yumeng ST:ADDRESS Wu Tong Road Number 366 ST:EMAIL 0623B01@zju.edu.cn ST:PHONE (0571)87236114 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - WT_1 Genotype:Wild-type | Sample source:Bone marrow derived macrophages RAW_FILE_NAME(Raw file name)=WT_1.mzML SUBJECT_SAMPLE_FACTORS - WT_2 Genotype:Wild-type | Sample source:Bone marrow derived macrophages RAW_FILE_NAME(Raw file name)=WT_2.mzML SUBJECT_SAMPLE_FACTORS - WT_3 Genotype:Wild-type | Sample source:Bone marrow derived macrophages RAW_FILE_NAME(Raw file name)=WT_3.mzML SUBJECT_SAMPLE_FACTORS - WT_4 Genotype:Wild-type | Sample source:Bone marrow derived macrophages RAW_FILE_NAME(Raw file name)=WT_4.mzML SUBJECT_SAMPLE_FACTORS - KO_1 Genotype:MG53-knockout | Sample source:Bone marrow derived macrophages RAW_FILE_NAME(Raw file name)=KO_1.mzML SUBJECT_SAMPLE_FACTORS - KO_2 Genotype:MG54-knockout | Sample source:Bone marrow derived macrophages RAW_FILE_NAME(Raw file name)=KO_2.mzML SUBJECT_SAMPLE_FACTORS - KO_3 Genotype:MG55-knockout | Sample source:Bone marrow derived macrophages RAW_FILE_NAME(Raw file name)=KO_3.mzML SUBJECT_SAMPLE_FACTORS - KO_4 Genotype:MG56-knockout | Sample source:Bone marrow derived macrophages RAW_FILE_NAME(Raw file name)=KO_4.mzML #COLLECTION CO:COLLECTION_SUMMARY Bone marrow-derived macrophages (BMDMs) were isolated from MG53-knockout (KO) CO:COLLECTION_SUMMARY mice and their wild-type (WT) littermates. After 5 days of in vitro culture, the CO:COLLECTION_SUMMARY culture supernatant was removed. Adherent BMDMs were then harvested by CO:COLLECTION_SUMMARY trypsinization and collected for subsequent metabolomic analysis. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY Bone marrow-derived macrophages (BMDMs) were isolated from MG53-knockout (KO) TR:TREATMENT_SUMMARY mice and their wild-type (WT) littermates. Cells were cultured in vitro for 5 TR:TREATMENT_SUMMARY days to allow for differentiation and stabilization, after which they were TR:TREATMENT_SUMMARY harvested for metabolomic analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Methanol (0.75 mL) was added to a 100 μL sample , which was placed into a glass SP:SAMPLEPREP_SUMMARY tube with a Teflon lined cap, and the tube was vortexed. 2.5 ml of MTBE was SP:SAMPLEPREP_SUMMARY added ， then 10 μL of SPLASH™ internal standard was added and the mixture SP:SAMPLEPREP_SUMMARY was incubated for 1 h at room temperature in a shaker. Phase separation was SP:SAMPLEPREP_SUMMARY induced by adding 0.625 ml of MS-grade water. Upon 10 min of incubation at room SP:SAMPLEPREP_SUMMARY temperature, the sample was centrifuged at 1,000 g for 10 min. The upper SP:SAMPLEPREP_SUMMARY (organic) phase was collected, and the lower phase was re-extracted with 1 mL of SP:SAMPLEPREP_SUMMARY the solvent mixture（MTBE/methanol/water (10:3:2.5, v/v/v)), and collecting the SP:SAMPLEPREP_SUMMARY upper phase. Combined organicphases were dried and dissolved in 100 μL of SP:SAMPLEPREP_SUMMARY isopropanol for storage. Then analyzed by LC-MS/MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish UHPLC CH:COLUMN_NAME Thermo Accucore C30 (150 x 2.1mm,2.6um) CH:SOLVENT_A acetonitrile / water (6/4) with 10 mM ammonium acetate and 0.1% formic acid CH:SOLVENT_B acetonitrile/ isopropanol (1/9) with 10 mM ammonium acetate and 0.1% formic acid CH:FLOW_GRADIENT 30-99% CH:FLOW_RATE 0.35mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Q ExactiveTM HF mass spectrometer was operated in positive [negative] polarity MS:MS_COMMENTS mode with sheath gas :40 psi, sweep gas: 0 L/min, auxiliary gasrate: 10 L/min [7 MS:MS_COMMENTS L/min], spray voltage: 3.5 kV, capillary temperature: 320◦C, heater MS:MS_COMMENTS temperature: 350℃, S-Lens RF level: 50, scan range: 114–1700 m/z, automatic MS:MS_COMMENTS gain control target: 3e6, normalized collision energy: 22eV; 24 eV; 28eV [22 MS:MS_COMMENTS eV；24 eV；28 eV], Injection time: 100 ms, Isolation window: 1m/z, automatic MS:MS_COMMENTS gaincontrol target（MS2）: 2e5, dynamic exclusion: 6s. MS:MS_RESULTS_FILE ST004306_AN007166_Results.txt UNITS:μg/g Has m/z:Yes Has RT:Yes RT units:Minutes #END