#METABOLOMICS WORKBENCH yazhouwang_20240816_001351 DATATRACK_ID:5111 STUDY_ID:ST003414 ANALYSIS_ID:AN005609 PROJECT_ID:PR002113 VERSION 1 CREATED_ON August 19, 2024, 7:07 pm #PROJECT PR:PROJECT_TITLE Metabolic analysis of Anterior Cingulate Cortex in two Autism Spectrum Disorder PR:PROJECT_TITLE mouse models PR:PROJECT_SUMMARY In this project, we performed metabolomic analysis of anterior cingulate cortex PR:PROJECT_SUMMARY (ACC) of two autism mouse models (Shank3b-/- and FMR1-/- mice), in comparison PR:PROJECT_SUMMARY with their corresponding wild type controls. The results showed abnormal PR:PROJECT_SUMMARY metabolism of amino acids, especially sulfur containing amino acid, in both PR:PROJECT_SUMMARY Shank3b-/- and FMR1-/- ACC. PR:INSTITUTE Fourth Military Medical University PR:LAST_NAME Wang PR:FIRST_NAME Yazhou PR:ADDRESS 169 Chang Le Xi Road, Xi'an, Shaanxi, 710032, China PR:EMAIL yazhouw@fmmu.edu.cn PR:PHONE +86-29-84774562 #STUDY ST:STUDY_TITLE Metabolic analysis of Anterior Cingulate Cortex in two Autism Spectrum Disorder ST:STUDY_TITLE mouse models ST:STUDY_SUMMARY In this project, we performed metabolomic analysis of anterior cingulate cortex ST:STUDY_SUMMARY (ACC) of two autism mouse models (Shank3b-/- and FMR1-/- mice), in comparison ST:STUDY_SUMMARY with their corresponding wild type controls. The results showed abnormal ST:STUDY_SUMMARY metabolism of amino acids, especially sulfur containing amino acid, in both ST:STUDY_SUMMARY Shank3b-/- and FMR1-/- ACC. ST:INSTITUTE Fourth Military Medical University ST:LAST_NAME Wang ST:FIRST_NAME Yazhou ST:ADDRESS 169 Chang Le Xi Road, Xi'an, Shaanxi, 710032, China ST:EMAIL yazhouw@fmmu.edu.cn ST:PHONE +86-29-84774562 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #FACTORS #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 ACC1 c57-1M Sample source:Anterior Cingulate Cortex | Treatment:WT | Genotype:C57 RAW_FILE_NAME=c57-1M.raw SUBJECT_SAMPLE_FACTORS ACC2 c57-2M Sample source:Anterior Cingulate Cortex | Treatment:WT | Genotype:C57 RAW_FILE_NAME=c57-2M.raw SUBJECT_SAMPLE_FACTORS ACC3 c57-3M Sample source:Anterior Cingulate Cortex | Treatment:WT | Genotype:C57 RAW_FILE_NAME=c57-3M.raw SUBJECT_SAMPLE_FACTORS ACC4 c57-4M Sample source:Anterior Cingulate Cortex | Treatment:WT | Genotype:C57 RAW_FILE_NAME=c57-4M.raw SUBJECT_SAMPLE_FACTORS ACC5 shank1M Sample source:Anterior Cingulate Cortex | Treatment:KO | Genotype:Shank3b-KO RAW_FILE_NAME=shank1M.raw SUBJECT_SAMPLE_FACTORS ACC6 shank2M Sample source:Anterior Cingulate Cortex | Treatment:KO | Genotype:Shank3b-KO RAW_FILE_NAME=shank2M.raw SUBJECT_SAMPLE_FACTORS ACC7 shank3M Sample source:Anterior Cingulate Cortex | Treatment:KO | Genotype:Shank3b-KO RAW_FILE_NAME=shank3M.raw SUBJECT_SAMPLE_FACTORS ACC8 shank4M Sample source:Anterior Cingulate Cortex | Treatment:KO | Genotype:Shank3b-KO RAW_FILE_NAME=shank4M.raw SUBJECT_SAMPLE_FACTORS ACC9 FVB1M Sample source:Anterior Cingulate Cortex | Treatment:WT | Genotype:FVB129 RAW_FILE_NAME=FVB1M.raw SUBJECT_SAMPLE_FACTORS ACC10 FVB2M Sample source:Anterior Cingulate Cortex | Treatment:WT | Genotype:FVB129 RAW_FILE_NAME=FVB2M.raw SUBJECT_SAMPLE_FACTORS ACC11 FVB3M Sample source:Anterior Cingulate Cortex | Treatment:WT | Genotype:FVB129 RAW_FILE_NAME=FVB3M.raw SUBJECT_SAMPLE_FACTORS ACC12 FVB4M Sample source:Anterior Cingulate Cortex | Treatment:WT | Genotype:FVB129 RAW_FILE_NAME=FVB4M.raw SUBJECT_SAMPLE_FACTORS ACC13 FMR1M Sample source:Anterior Cingulate Cortex | Treatment:KO | Genotype:FMR1-KO RAW_FILE_NAME=FMR1M.raw SUBJECT_SAMPLE_FACTORS ACC14 FMR2M Sample source:Anterior Cingulate Cortex | Treatment:KO | Genotype:FMR1-KO RAW_FILE_NAME=FMR2M.raw SUBJECT_SAMPLE_FACTORS ACC15 FMR3M Sample source:Anterior Cingulate Cortex | Treatment:KO | Genotype:FMR1-KO RAW_FILE_NAME=FMR3M.raw SUBJECT_SAMPLE_FACTORS ACC16 FMR4M Sample source:Anterior Cingulate Cortex | Treatment:KO | Genotype:FMR1-KO RAW_FILE_NAME=FMR4M.raw #COLLECTION CO:COLLECTION_SUMMARY Tissues were quickly isolated and individually grounded with liquid nitrogen. CO:SAMPLE_TYPE Brain cortex #TREATMENT TR:TREATMENT_SUMMARY WT and KO mice were used. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Tissues were individually grounded with liquid nitrogen and the homogenate was SP:SAMPLEPREP_SUMMARY resuspended with 500μL prechilled 80% methanol by well vortex. The samples were SP:SAMPLEPREP_SUMMARY incubated on ice for 5 min and then were centrifuged at 15,000 g, 4°C for 20 SP:SAMPLEPREP_SUMMARY min. Some of supernatant was diluted to final concentration containing 53% SP:SAMPLEPREP_SUMMARY methanol by LC-MS grade water. The samples were subsequently transferred to a SP:SAMPLEPREP_SUMMARY fresh Eppendorf tube and then were centrifuged at 15000 g, 4°C for 20 min. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Vanquish UHPLC CH:COLUMN_NAME Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0 min, 1% B; 1 min, 1% B; 8 min, 99% B; 10 min, 99% B; 10.1 min, 1% B; 12 min, CH:FLOW_GRADIENT 1% B. CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 40° C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The acquisition software (Xcalibur 4.0.27, Thermo) was used to evaluates the MS:MS_COMMENTS full scan survey MS data.ESI source conditions were set as follows: sheath gas MS:MS_COMMENTS flow rate was 45 Arb, aux gas flow rate was 15 Arb, capillary temperature was MS:MS_COMMENTS 320° C, full MS resolution was 70,000, MS/MS resolution was 17,500, collision MS:MS_COMMENTS energy was 20/40/60 eV in the NCE model, spray voltage was 3.8 kV (positive) or MS:MS_COMMENTS −3.1 kV (negative), respectively. The peak identification was performed as MS:MS_COMMENTS follows: The raw data files generated by UHPLC-MS/MS were processed using the MS:MS_COMMENTS Compound Discoverer 3.1 (CD3.1, Thermo Fisher) to perform peak alignment, peak MS:MS_COMMENTS picking, and quantitation for each metabolite. The main parameters were set as MS:MS_COMMENTS follows: retention time tolerance, 0.2minutes actual mass tolerance, 5ppm signal MS:MS_COMMENTS intensity tolerance, 30% signal/noise ratio, 3 and minimum intensity, 100,000. MS:MS_COMMENTS After that, peak intensities were normalized to the total spectral intensity. MS:MS_COMMENTS The normalized data was used to predict the molecular formula based on additive MS:MS_COMMENTS ions, molecular ion peaks and fragment ions. And then peaks were matched with MS:MS_COMMENTS the mzCloud(https://www.mzcloud.org/)， mz Vaultand Mass Listdatabase to obtain MS:MS_COMMENTS the accurate qualitative and relative quantitative results. Statistical analyses MS:MS_COMMENTS were performed using the statistical software R (R version R-3.4.3), Python MS:MS_COMMENTS (Python 2.7.6 version) and CentOS (CentOS release 6.6) MS:MS_RESULTS_FILE ST003414_AN005609_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END