#METABOLOMICS WORKBENCH bigfatgoalie01_20231005_101400 DATATRACK_ID:4377 STUDY_ID:ST002930 ANALYSIS_ID:AN004808 PROJECT_ID:PR001822 VERSION 1 CREATED_ON October 11, 2023, 2:16 pm #PROJECT PR:PROJECT_TITLE Role of hypoxia inducible factor-1a (HIF1a) in Skeletal Muscle Physiology PR:PROJECT_SUMMARY Hypoxia-inducible factor (HIF)-1a is continuously synthesized and degraded in PR:PROJECT_SUMMARY normoxia, whereas during hypoxia, HIF1a stabilization restricts cellular oxygen PR:PROJECT_SUMMARY utilization. Less is known about HIF1a function(s) and sex-specific effects PR:PROJECT_SUMMARY during normoxia in the basal state. Since skeletal muscle is the largest protein PR:PROJECT_SUMMARY store in mammals and protein homeostasis has high energy demands, we determined PR:PROJECT_SUMMARY HIF1a function at baseline during normoxia in skeletal muscle with the use of PR:PROJECT_SUMMARY untargeted metabolomics. PR:INSTITUTE Cleveland Clinic PR:DEPARTMENT Inflammation and Immunity PR:LABORATORY Dasarathy Lab PR:LAST_NAME Dasarathy PR:FIRST_NAME Srinivasan PR:ADDRESS 9500 Euclid Avenue PR:EMAIL dasaras@ccf.org PR:PHONE 2163799846 PR:FUNDING_SOURCE NIH #STUDY ST:STUDY_TITLE Role of Hypoxia-inducible factor-1a (HIF1a)in Skeletal Muscle Physiology (C2C12 ST:STUDY_TITLE myotube model) ST:STUDY_SUMMARY Hypoxia-inducible factor (HIF)-1a is continuously synthesized and degraded in ST:STUDY_SUMMARY normoxia, whereas during hypoxia, HIF1a stabilization restricts cellular oxygen ST:STUDY_SUMMARY utilization. Less is known about HIF1a function(s) and sex-specific effects ST:STUDY_SUMMARY during normoxia in the basal state. Since skeletal muscle is the largest protein ST:STUDY_SUMMARY store in mammals and protein homeostasis has high energy demands, we determined ST:STUDY_SUMMARY HIF1a function at baseline during normoxia in C2C12 murine myotubes with the use ST:STUDY_SUMMARY of untargeted metabolomics. 114 samples of extracted metabolites from cells were ST:STUDY_SUMMARY analyzed using LCMS. We identified that metabolites, especially those in the ST:STUDY_SUMMARY glycolytic pathway and TCA cycle, were differentially expressed in cells with ST:STUDY_SUMMARY HIF1a KO compared to WT. ST:INSTITUTE Cleveland Clinic ST:DEPARTMENT Inflamation and Immunity ST:LABORATORY Dasarathy Lab ST:LAST_NAME Dasarathy ST:FIRST_NAME Srinivasan ST:ADDRESS 9500 Euclid Avenue ST:EMAIL dasaras@ccf.org ST:PHONE 2163799846 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE Passage 4-8 SU:GENDER Female #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 C2C12 unit1 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG189 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 unit1 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG189 SUBJECT_SAMPLE_FACTORS - WT C2C12 unit2 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG190 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 unit2 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG190 SUBJECT_SAMPLE_FACTORS - WT C2C12 unit3 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG191 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 unit3 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG191 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT4 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG201 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 UnT4 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG201 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT5 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG202 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 UnT5 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG202 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT6 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG203 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 UnT6 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG203 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT7 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG204 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 UnT7 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG204 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT8 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG205 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 UnT8 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG205 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT9 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG206 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=WT C2C12 UnT9 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG206 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT1 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG249 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=HIF KO C2C12 UnT1 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG249 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT2 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG250 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=HIF KO C2C12 UnT2 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG250 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT3 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG251 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=HIF KO C2C12 UnT3 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG251 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT4 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG252 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=HIF KO C2C12 UnT4 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG252 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT5 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG253 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=HIF KO C2C12 UnT5 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG253 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT6 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG254 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Negative; RAW_FILE_NAME=HIF KO C2C12 UnT6 M_Dasarathy_Nicole_114Cells_02042022_HILIC_NEG254 SUBJECT_SAMPLE_FACTORS - WT C2C12 unit1 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS189 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 unit1 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS189 SUBJECT_SAMPLE_FACTORS - WT C2C12 unit2 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS190 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 unit2 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS190 SUBJECT_SAMPLE_FACTORS - WT C2C12 unit3 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS191 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 unit3 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS191 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT4 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS201 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 UnT4 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS201 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT5 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS202 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 UnT5 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS202 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT6 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS203 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 UnT6 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS203 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT7 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS204 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 UnT7 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS204 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT8 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS205 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 UnT8 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS205 SUBJECT_SAMPLE_FACTORS - WT C2C12 UnT9 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS206 Genotype:Wild-type | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=WT C2C12 UnT9 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS206 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT1 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS249 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=HIF KO C2C12 UnT1 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS249 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT2 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS250 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=HIF KO C2C12 UnT2 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS250 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT3 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS251 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=HIF KO C2C12 UnT3 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS251 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT4 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS252 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=HIF KO C2C12 UnT4 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS252 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT5 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS253 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=HIF KO C2C12 UnT5 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS253 SUBJECT_SAMPLE_FACTORS - HIF KO C2C12 UnT6 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS254 Genotype:HIF1a KO | Treatment:Untreated Column=HILIC; Mode=Positive; RAW_FILE_NAME=HIF KO C2C12 UnT6 M_Dasarathy_Nicole_114Cells_02042022_HILIC_POS254 #COLLECTION CO:COLLECTION_SUMMARY Metabolites were extracted from myotubes and skeletal muscle using chilled CO:COLLECTION_SUMMARY methanol containing 6 labeled internal standards (Betaine-d9, Carnitine-d9, CO:COLLECTION_SUMMARY Estrone-13C3, Cholesterol-13C3, Valeric acid-d9, Choline-d13). After CO:COLLECTION_SUMMARY centrifugation at 14,000g for 20 minutes to precipitate the protein pellet, the CO:COLLECTION_SUMMARY supernatant was removed, dried, and reconstituted in 5% acetonitrile. LC/MS CO:COLLECTION_SUMMARY (liquid chromatography/mass spectrometry) analysis was then performed, including CO:COLLECTION_SUMMARY on pooled quality control samples. CO:SAMPLE_TYPE Skeletal myotubes #TREATMENT TR:TREATMENT_SUMMARY Cells were not treated (Untreated, UnT) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from myotubes and skeletal muscle using chilled SP:SAMPLEPREP_SUMMARY methanol containing 6 labeled internal standards (Betaine-d9, Carnitine-d9, SP:SAMPLEPREP_SUMMARY Estrone-13C3, Cholesterol-13C3, Valeric acid-d9, Choline-d13). After SP:SAMPLEPREP_SUMMARY centrifugation at 14,000g for 20 minutes to precipitate the protein pellet, the SP:SAMPLEPREP_SUMMARY supernatant was removed, dried, and reconstituted in 5% acetonitrile. LC/MS SP:SAMPLEPREP_SUMMARY (liquid chromatography/mass spectrometry) analysis was then performed, including SP:SAMPLEPREP_SUMMARY on pooled quality control samples. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) CH:SOLVENT_A 100% water;10 mM ammonium acetate; 0.125% acetic acid CH:SOLVENT_B 5% water/95% acetonitrile;10 mM ammonium acetate; 0.125% acetic acid CH:FLOW_GRADIENT Time (min.) Flow Rate (mL/min) %B 0.0 0.2 100 2.0 0.2 70 4.0 0.2 60 5.5 0.2 60 CH:FLOW_GRADIENT 10.5 0.2 50 11.5 0.2 50 12.0 0.2 100 16.0 0.2 100 CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Data dependent acquisitions (DDA) on the pooled representative QC samples MS:MS_COMMENTS include MS full scans at a resolution of 120,000 and HCD MS/MS scans taken on MS:MS_COMMENTS the top 5 most abundant ions at a resolution of 30,000 with dynamic exclusion. MS:MS_COMMENTS The resolution of the MS2 scans were taken at a stepped NCE energy of 10.0, 20.0 MS:MS_COMMENTS and 30.0. MS:MS_RESULTS_FILE ST002930_AN004808_Results.txt UNITS:relative abundance Has m/z:Yes Has RT:Yes RT units:Seconds #END