#METABOLOMICS WORKBENCH matser_20250120_135229 DATATRACK_ID:5546 STUDY_ID:ST003689 ANALYSIS_ID:AN006054 PROJECT_ID:PR002288
VERSION             	1
CREATED_ON             	January 27, 2025, 5:49 pm
#PROJECT
PR:PROJECT_TITLE                 	Effect of pyruvate dehydrogenase kinase 1 (PDK1) on H9c2 cardiomyocyte
PR:PROJECT_TITLE                 	metabolism
PR:PROJECT_SUMMARY               	Pyruvate dehydrogenase kinases are known to be stimulated during fasted
PR:PROJECT_SUMMARY               	conditions. To test the specific role of PDK1 in cardiomyocyte metabolism, PDK1
PR:PROJECT_SUMMARY               	was knocked down in H9c2 cells using lentiviral shRNA. Cells were also
PR:PROJECT_SUMMARY               	transduced with a scrambled shRNA for controls. Cells were seeded in complete
PR:PROJECT_SUMMARY               	growth media (high glucose DMEM with 10% FBS) containing palmitate for 18 h to
PR:PROJECT_SUMMARY               	stimulate lipid accumulation. Then, transferred to nutrient-depleted media (low
PR:PROJECT_SUMMARY               	glucose DMEM, no FBS) to stimulate nutrient turnover. The metabolome (study
PR:PROJECT_SUMMARY               	#5546) and lipidome (study #5545) were analyzed in an end-time study and time
PR:PROJECT_SUMMARY               	series study respectively.
PR:INSTITUTE                     	University of British Columbia
PR:LAST_NAME                     	Atser
PR:FIRST_NAME                    	Michael
PR:ADDRESS                       	2350 Health Sciences Mall
PR:EMAIL                         	matser@student.ubc.ca
PR:PHONE                         	+16042193298
#STUDY
ST:STUDY_TITLE                   	Role of pyruvate dehydrogenase kinase 1 (PDK1) in glucose metabolism in H9c2
ST:STUDY_TITLE                   	cardiomyocyte cells
ST:STUDY_SUMMARY                 	Pyruvate dehydrogenase kinase 1 (PDK1) belongs to a family of enzymes that
ST:STUDY_SUMMARY                 	inhibit pyruvate dehydrogenase consequently inhibiting glucose oxidation. In
ST:STUDY_SUMMARY                 	cardiomyocytes, total PDK activity is elevated during nutrient depleted
ST:STUDY_SUMMARY                 	conditions to phosphorylate pyruvate dehydrogenase, resulting in the inhibition
ST:STUDY_SUMMARY                 	of the oxidative decarboxylation of pyruvate to acetyl-CoA, critical to glucose
ST:STUDY_SUMMARY                 	oxidation. However, the contributing role of PDK1 to this overall effect is
ST:STUDY_SUMMARY                 	unclear in cardiomyocytes. So, we designed an end-point study to test out the
ST:STUDY_SUMMARY                 	specific role of PDK1 in cardiomyocyte glucose metabolism. We tested the impact
ST:STUDY_SUMMARY                 	of PDK1 on cardiomyocyte glucose metabolism. We utilized uniformly labeled
ST:STUDY_SUMMARY                 	glucose to better trace glucose metabolism. Briefly, PDK1 knockdown and control
ST:STUDY_SUMMARY                 	H9c2 cells were first differentiated into a cardiomyocyte phenotype then treated
ST:STUDY_SUMMARY                 	with palmitic acid then nutrient-deprived in in media containing 13C6 glucose.
ST:STUDY_SUMMARY                 	Parallel experiments were performed by treating cells with palmitate and
ST:STUDY_SUMMARY                 	incubating in regular/unlabelled nutrient-depleted media for 3 h. Metabolomics
ST:STUDY_SUMMARY                 	analysis was performed on collected cells.
ST:INSTITUTE                     	University of British Columbia
ST:DEPARTMENT                    	Cell and Physiological Sciences
ST:LABORATORY                    	Johnson Lab
ST:LAST_NAME                     	Atser
ST:FIRST_NAME                    	Michael
ST:ADDRESS                       	2350 Health Sciences Mall
ST:EMAIL                         	matser@student.ubc.ca
ST:PHONE                         	+16042193298
ST:NUM_GROUPS                    	4
ST:TOTAL_SUBJECTS                	12
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Rattus norvegicus
SU:TAXONOMY_ID                   	10116
SU:CELL_BIOSOURCE_OR_SUPPLIER    	Millipore Sigma
SU:CELL_COUNTS                   	400,000 cells
SU:SPECIES_GROUP                 	Mammalian
#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           	-	Lenti-C_13C_1 hour_Expt I	Sample source:H9c2 cell line | shRNA:pdk1 shRNA | Glucose labeling:labelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-C_13C_1 hour_Expt I_10-24-2023_R-C6_14764.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-C_13C_1 hour_Expt II	Sample source:H9c2 cell line | shRNA:pdk1 shRNA | Glucose labeling:labelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-C_13C_1 hour_Expt II_10-24-2023_R-D3_14757.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-C_13C_1 hour_Expt III	Sample source:H9c2 cell line | shRNA:pdk1 shRNA | Glucose labeling:labelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-C_13C_1 hour_Expt III_10-25-2023_R-D9_14800.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-C_3 hour_Expt I	Sample source:H9c2 cell line | shRNA:pdk1 shRNA | Glucose labeling:unlabelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-C_3 hour_Expt I_10-25-2023_R-E4_14792.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-C_3 hour_Expt II	Sample source:H9c2 cell line | shRNA:pdk1 shRNA | Glucose labeling:unlabelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-C_3 hour_Expt II_10-25-2023_R-E5_14776.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-C_3 hour_Expt III	Sample source:H9c2 cell line | shRNA:pdk1 shRNA | Glucose labeling:unlabelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-C_3 hour_Expt III_10-25-2023_R-E6_14794.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-SC_13C_1 hour_Expt I	Sample source:H9c2 cell line | shRNA:scrambled shRNA | Glucose labeling:labelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-SC_13C_1 hour_Expt I_10-25-2023_R-A6_14784.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-SC_13C_1 hour_Expt II	Sample source:H9c2 cell line | shRNA:scrambled shRNA | Glucose labeling:labelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-SC_13C_1 hour_Expt II_10-24-2023_R-B3_14774.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-SC_13C_1 hour_Expt III	Sample source:H9c2 cell line | shRNA:scrambled shRNA | Glucose labeling:labelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-SC_13C_1 hour_Expt III_10-25-2023_R-B9_14783.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-SC_3 hour_Expt I	Sample source:H9c2 cell line | shRNA:scrambled shRNA | Glucose labeling:unlabelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-SC_3 hour_Expt I_10-25-2023_R-E1_14782.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-SC_3 hour_Expt II	Sample source:H9c2 cell line | shRNA:scrambled shRNA | Glucose labeling:unlabelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-SC_3 hour_Expt II_10-25-2023_R-E2_14786.mzML
SUBJECT_SAMPLE_FACTORS           	-	Lenti-SC_3 hour_Expt III	Sample source:H9c2 cell line | shRNA:scrambled shRNA | Glucose labeling:unlabelled	RAW_FILE_NAME(Raw file name Metabolomics POS)=Lenti-SC_3 hour_Expt III_10-25-2023_R-E3_14778.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC2	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=QC2_10-24-2023_R-F8_14760.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC3	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=QC3_10-24-2023_R-F8_14771.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC4	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=QC4_10-25-2023_R-F8_14780.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC5	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=QC5_10-25-2023_R-F8_14791.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC0	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=QC0_10-24-2023_R-F8_14745.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC1	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=QC1_10-24-2023_R-F8_14750.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK2	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=BLK2_10-24-2023_R-F9_14759.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK3	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=BLK3_10-24-2023_R-F9_14769.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK4	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=BLK4_10-25-2023_R-F9_14789.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK5	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=BLK5_10-25-2023_R-F9_14802.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK0	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=BLK0_10-23-2023_R-F9_14671.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK6	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=BLK6_10-24-2023_R-F9_14744.mzML
SUBJECT_SAMPLE_FACTORS           	-	BLK1	Sample source:- | shRNA:- | Glucose labeling:-	RAW_FILE_NAME(Raw file name Metabolomics POS)=BLK1_10-24-2023_R-F9_14746.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	H9c2 were transduced with lentiviral Pdk1 shRNA to knockdown PDK1. Control cells
CO:COLLECTION_SUMMARY            	were transduced with a scrambled shRNA. Cells were then differentiated into a
CO:COLLECTION_SUMMARY            	cardiomyocyte phenotype by maintaining in DMEM + 1% FBS + 1 micro molar
CO:COLLECTION_SUMMARY            	retinoic-acid for 10 days. Following experimental treatment, cells were washed
CO:COLLECTION_SUMMARY            	with ice-cold phosphate-buffered saline (PBS), scraped on ice, centrifuged at
CO:COLLECTION_SUMMARY            	max speed at 4 °C for 5 mins, and pellet stored at -80 °C.
CO:SAMPLE_TYPE                   	H9c2 cells
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Differentiated PDK1 knockdown and control cells were first treated with 0.15 mM
TR:TREATMENT_SUMMARY             	palmitic acid complexed to bovine serum albumin (6:1 molar ratio) for 18 h, then
TR:TREATMENT_SUMMARY             	transferred to nutrient-depleted media (low glucose(5.5 mM unlabelled glucose)
TR:TREATMENT_SUMMARY             	DMEM) for 2 h. To label cells with 13C6 glucose, cells were then transferred to
TR:TREATMENT_SUMMARY             	DMEM containing 5.5 mM 13C6 glucose for an additional 1 h, bringing the total
TR:TREATMENT_SUMMARY             	time in nutrient-depleted media to 3 h. Parallel experiments were performed by
TR:TREATMENT_SUMMARY             	treating cells with palmitate and incubating in regular/unlabelled
TR:TREATMENT_SUMMARY             	nutrient-depleted media for 3 h. After the 3 h nutrient-depletion, cells were
TR:TREATMENT_SUMMARY             	washed with ice-cold phosphate buffered saline (PBS), scraped on ice,
TR:TREATMENT_SUMMARY             	centrifuged at max speed at 4 °C for 5 mins, and pellet stored at -80 °C.
TR:CELL_MEDIA                    	Dulbecco's Modified Eagle Medium
TR:CELL_HARVESTING               	Cells were washed with ice-cold phosphate buffered saline (PBS), scraped on ice,
TR:CELL_HARVESTING               	centrifuged at max speed at 4 °C for 5 mins
TR:CELL_PCT_CONFLUENCE           	70%
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Samples were analyzed following previously established protocols with some
SP:SAMPLEPREP_SUMMARY            	modifications (Black, Braydon, Leandro Buffoni Roque da Silva, Guanggan Hu,
SP:SAMPLEPREP_SUMMARY            	Xianya Qu, Daniel FQ Smith, Armando Alcázar Magaña, Linda C. Horianopoulos et
SP:SAMPLEPREP_SUMMARY            	al. "Glutathione-mediated redox regulation in Cryptococcus neoformans impacts
SP:SAMPLEPREP_SUMMARY            	virulence." Nature microbiology 9, no. 8 (2024): 2084-2098.
SP:SAMPLEPREP_SUMMARY            	https://doi.org/10.1038/s41564-024-01721-x ; McAfee, Alison, Armando Alcazar
SP:SAMPLEPREP_SUMMARY            	Magana, Leonard James Foster, and Shelley E. Hoover. "Parallel pheromone,
SP:SAMPLEPREP_SUMMARY            	metabolite, and lipid analyses reveal patterns associated with early life
SP:SAMPLEPREP_SUMMARY            	transitions and ovary activation in honey bee (Apis mellifera) queens." bioRxiv
SP:SAMPLEPREP_SUMMARY            	(2024): 2024-04. https://doi.org/10.1101/2024.04.19.590367). The polar fraction
SP:SAMPLEPREP_SUMMARY            	was resuspended in 200 µL of 50% aqueous methanol (v/v) spiked with internal
SP:SAMPLEPREP_SUMMARY            	standards at 1 ppm (methionine-d3, ferulic acid-d3, caffeine-13C3). Following
SP:SAMPLEPREP_SUMMARY            	resuspension, the mixture was centrifuged at 14,000 rcf for 10 minutes, and 150
SP:SAMPLEPREP_SUMMARY            	µL of the resulting supernatant was transferred to LC-MS vials (Thermo Fisher)
SP:SAMPLEPREP_SUMMARY            	for subsequent LC-MS/MS analysis. To assess analytical performance, 20 µL
SP:SAMPLEPREP_SUMMARY            	aliquots from each sample were pooled to create a quality control (QC) sample.
SP:PROCESSING_STORAGE_CONDITIONS 	4℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Separation of compounds was performed using a multigradient method on an
CH:CHROMATOGRAPHY_SUMMARY        	Inertsil Ph-3 UHPLC column (2 µm, 150 x 2.1 mm, GL Sciences), which was
CH:CHROMATOGRAPHY_SUMMARY        	equipped with a Ph-3 guard column (2 µm, 2.1 x 10 mm). The mobile phase
CH:CHROMATOGRAPHY_SUMMARY        	consisted of water (A) with 0.1% (v/v) formic acid and methanol (B) with 0.1%
CH:CHROMATOGRAPHY_SUMMARY        	(v/v) formic acid. Chromatographic separation was achieved using a multi-step
CH:CHROMATOGRAPHY_SUMMARY        	gradient from 5% to 99% mobile phase B over 18 minutes. The UHPLC program was
CH:CHROMATOGRAPHY_SUMMARY        	set as follows: 0 min (5% B); 0–1 min (5% B); 1–8 min (35% B); 8–10.5 min
CH:CHROMATOGRAPHY_SUMMARY        	(99% B); 10.5–14 min (99% B); 14–14.5 min (5% B); 14.5–18 min (5% B). The
CH:CHROMATOGRAPHY_SUMMARY        	column temperature was maintained at 55°C, with the autosampler kept at 4°C,
CH:CHROMATOGRAPHY_SUMMARY        	and a flow rate of 0.3 mL/min was employed - 5 µL injected in both positive and
CH:CHROMATOGRAPHY_SUMMARY        	negative ionization modes.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Inertsil Ph-3 (150 x 2.1mm, 2um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% methanol; 0.1% formic acid
CH:FLOW_GRADIENT                 	0 min (5% B); 0–1 min (5% B); 1–8 min (35% B); 8–10.5 min (99% B);
CH:FLOW_GRADIENT                 	10.5–14 min (99% B); 14–14.5 min (5% B); 14.5–18 min (5% B)
CH:FLOW_RATE                     	0.3mL/min
CH:COLUMN_TEMPERATURE            	55
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Bruker Impact II
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Data-dependent acquisitions were performed in positive (ESI+) ionization modes,
MS:MS_COMMENTS                   	to capture precursor and fragment ions for compound annotation. For ESI+, the
MS:MS_COMMENTS                   	mass spectrometer parameters were as follows: capillary voltage of 4,500 V,
MS:MS_COMMENTS                   	nebulizer gas pressure of 2.0 bar, dry gas flow rate of 9 L/min, dry gas
MS:MS_COMMENTS                   	temperature of 220°C, mass scan range of 60–1,300 m/z, and a total cycle time
MS:MS_COMMENTS                   	of 0.6 s. To ensure comprehensive structural information, collision energy of 20
MS:MS_COMMENTS                   	V was ramped from 100% to 250% during each MS/MS scan. High mass accuracy was
MS:MS_COMMENTS                   	maintained by performing internal calibration during each analytical run using
MS:MS_COMMENTS                   	10 µL of 10 mM sodium formate, injected at the start of the run (from 0 to 0.15
MS:MS_COMMENTS                   	min) via a 6-port valve.
MS:CAPILLARY_VOLTAGE             	4500 v
MS:MS_RESULTS_FILE               	ST003689_AN006054_Results.txt	UNITS:Peak Area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END