#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