#METABOLOMICS WORKBENCH erinzhang_0220_20211027_143938 DATATRACK_ID:2910 STUDY_ID:ST001959 ANALYSIS_ID:AN003194 VERSION 1 CREATED_ON 03-03-2022 #PROJECT PR:PROJECT_TITLE Serum metabolomics study in intestinal AMPK KO mice PR:PROJECT_TYPE Serum untargeted metabolomics PR:PROJECT_SUMMARY Serum untargeted metabolomics studies on serum samples from intestinal AMPK KO PR:PROJECT_SUMMARY mice and control mice PR:INSTITUTE City of Hope National Medical Center PR:DEPARTMENT Diabetes Complications & Metabolism PR:LAST_NAME Zhang PR:FIRST_NAME Eryun PR:ADDRESS 1500 Duarte Rd, Duarte, CA, 91010, USA PR:EMAIL erzhang@coh.org PR:PHONE 6268735517 PR:DOI http://dx.doi.org/10.21228/M8NM5W #STUDY ST:STUDY_TITLE Investigation of serum metabolites in the AMPK intestinal KO mice ST:STUDY_SUMMARY Conducted serum untargeted metabolomics analysis in AMP-activated protein kinase ST:STUDY_SUMMARY (AMPK) intestinal KO mice and control mice under high-fat diet (HFD) conditions ST:INSTITUTE City of Hope National Medical Center ST:DEPARTMENT Diabetes Complications & Metabolism ST:LAST_NAME Zhang ST:FIRST_NAME Eryun ST:ADDRESS 1500 Duarte Rd, Duarte, CA91010 ST:EMAIL zhangey0220@126.com ST:PHONE 16268735517 ST:SUBMIT_DATE 2021-10-27 #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]Additional sample data SUBJECT_SAMPLE_FACTORS - A328pos Genotype:AMPKfl/fl(control) mice | Treatment:HFD RAW_FILE_NAME=A328pos.mzXML SUBJECT_SAMPLE_FACTORS - A329pos Genotype:AMPKfl/fl(control) mice | Treatment:HFD RAW_FILE_NAME=A329pos.mzXML SUBJECT_SAMPLE_FACTORS - A330pos Genotype:AMPKfl/fl(control) mice | Treatment:HFD RAW_FILE_NAME=A330pos.mzXML SUBJECT_SAMPLE_FACTORS - A331pos Genotype:AMPKfl/fl(control) mice | Treatment:HFD RAW_FILE_NAME=A331pos.mzXML SUBJECT_SAMPLE_FACTORS - B332pos Genotype:AMPK intestinal KO mice | Treatment:HFD RAW_FILE_NAME=B332pos.mzXML SUBJECT_SAMPLE_FACTORS - B333pos Genotype:AMPK intestinal KO mice | Treatment:HFD RAW_FILE_NAME=B333pos.mzXML SUBJECT_SAMPLE_FACTORS - B334pos Genotype:AMPK intestinal KO mice | Treatment:HFD RAW_FILE_NAME=B334pos.mzXML SUBJECT_SAMPLE_FACTORS - B335pos Genotype:AMPK intestinal KO mice | Treatment:HFD RAW_FILE_NAME=B335pos.mzXML #COLLECTION CO:COLLECTION_SUMMARY Collect whole blood in a covered test tube,leave the tube in a standing position CO:COLLECTION_SUMMARY and wait 30 min.Centrifuge 1500 g 10 min at 4ºC.Take out the serum. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY C57BL/6-AMPKa1flox/flox mice (#014141), and B6.Cg-Tg (Vil-cre) 1000Gum/J TR:TREATMENT_SUMMARY (#021504) were purchased from Jackson Laboratory (Bar Harbor, ME). AMPK TR:TREATMENT_SUMMARY intestinal KO (AMPK-IKO) mice were generated by cross-breeding TR:TREATMENT_SUMMARY C57BL/6-AMPKa1flox/flox mice with B6.Cg-Tg (Vil-cre) 1000Gum/J. All animal TR:TREATMENT_SUMMARY procedures were approved by the City of Hope Institutional Animal Care and Use TR:TREATMENT_SUMMARY Committee and conducted in accordance with the National Institutes of Health TR:TREATMENT_SUMMARY Guidelines for the Care and Use of Laboratory Animals. 6-week-old male mice were TR:TREATMENT_SUMMARY housed in a temperature (22-23°C) and light-controlled vivarium with free TR:TREATMENT_SUMMARY access to water and high-fat diet (HFD, 60% kcal fat; D12492, Research Diets, TR:TREATMENT_SUMMARY New Brunswick, NJ) for 12 weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 50 µL of serum was used for metabolites extraction. 200 μL extract solution SP:SAMPLEPREP_SUMMARY (acetonitrile: methanol = 1: 1) containing isotopically labeled internal SP:SAMPLEPREP_SUMMARY standard mixture was added to each sample. After vortexing for 1 min, the SP:SAMPLEPREP_SUMMARY samples were sonicated for 10 min in cold water bath. Then, the samples were SP:SAMPLEPREP_SUMMARY incubated at -40 °C for 1 h, and centrifuged at 12000 rpm for 15 min at 4°C. SP:SAMPLEPREP_SUMMARY 200 µL of supernatant were transferred to a fresh tube, and dried in a vacuum SP:SAMPLEPREP_SUMMARY concentrator at 37 °C. Then, the dried samples were reconstituted in 200 µL of SP:SAMPLEPREP_SUMMARY 50% acetonitrile by sonication on ice for 10 min. The constitution was then SP:SAMPLEPREP_SUMMARY centrifuged at 13000 rpm for 15 min at 4°C, and 75 µL of supernatant were SP:SAMPLEPREP_SUMMARY transferred to a fresh glass vial for LC/MS analysis. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5600 TripleTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS UHPLC-MS raw data files were converted into mzXML format using the MS:MS_COMMENTS “msconvert” program from Pro-teoWizard and then analyzed by the XCMS 67 and MS:MS_COMMENTS CAMERA toolbox 68 with R statistical software. The CentWave algorithm in XCMS MS:MS_COMMENTS was used for peak detection. The parameter “peak-width” was set as (5, 20) MS:MS_COMMENTS in units of seconds, referring to the minimum and maximum peak widths for peak MS:MS_COMMENTS detection. The pa-rameter ‘‘snthresh’’ is set as 3 for sensitive peak MS:MS_COMMENTS detection. For multiple UHPLC–MS data files, an or-dered bijective MS:MS_COMMENTS interpolated warping (OBI-Warp) algorithm in XCMS was used for peak alignment. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST001959_AN003194_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END