#METABOLOMICS WORKBENCH Karin_20241006_114144 DATATRACK_ID:5264 STUDY_ID:ST003544 ANALYSIS_ID:AN005821 PROJECT_ID:PR002180
VERSION             	1
CREATED_ON             	October 9, 2024, 2:10 am
#PROJECT
PR:PROJECT_TITLE                 	Metabolomics of Ndufs4 KO human induced pluripotent stem cells (iPSCs)
PR:PROJECT_TYPE                  	Multi-platform metabolomics analysis
PR:PROJECT_SUMMARY               	Mitochondrial diseases, often linked to complex I (CI) defects, lack curative
PR:PROJECT_SUMMARY               	treatments. High-throughput drug screening using human-relevant platforms is
PR:PROJECT_SUMMARY               	crucial for identifying new therapeutics. Induced pluripotent stem cell (iPSC)
PR:PROJECT_SUMMARY               	and CRISPR technologies offer a powerful tool for this purpose. While typically
PR:PROJECT_SUMMARY               	differentiated into disease-relevant cell types, recent studies support the use
PR:PROJECT_SUMMARY               	of undifferentiated iPSCs for drug discovery. The aim of this project was to
PR:PROJECT_SUMMARY               	develop and characterize NDUFS4 KO iPSCs in their pluripotent state. The
PR:PROJECT_SUMMARY               	metabolic profile of Ndufs4 KO human induced pluripotent stem cells (iPSCs) were
PR:PROJECT_SUMMARY               	compared to that of isogenic controls using multi-platform metabolomics,
PR:PROJECT_SUMMARY               	consisting of targeted LC-MS/MS, targeted GC-MS/MS, and untargeted GC-TOFMS
PR:PROJECT_SUMMARY               	analyses. Metabolic profiling revealed a distinct phenotype in NDUFS4 KO iPSCs,
PR:PROJECT_SUMMARY               	predominantly associated with an elevated NADH/NAD+ ratio, consistent with
PR:PROJECT_SUMMARY               	alterations observed in other models of mitochondrial dysfunction. These
PR:PROJECT_SUMMARY               	findings underscore the potential of iPSCs for early-stage, high-throughput
PR:PROJECT_SUMMARY               	therapeutic screening in mitochondrial diseases.
PR:INSTITUTE                     	North-West University
PR:LAST_NAME                     	Louw
PR:FIRST_NAME                    	Roan
PR:ADDRESS                       	Hofman Street, Potchefstroom, North-West, 2520, South Africa
PR:EMAIL                         	Roan.Louw@nwu.ac.za
PR:PHONE                         	+27182994074
#STUDY
ST:STUDY_TITLE                   	Metabolomics of Ndufs4 KO human induced pluripotent stem cells (iPSCs)
ST:STUDY_SUMMARY                 	Mitochondrial diseases, often linked to complex I (CI) defects, lack curative
ST:STUDY_SUMMARY                 	treatments. High-throughput drug screening using human-relevant platforms is
ST:STUDY_SUMMARY                 	crucial for identifying new therapeutics. Induced pluripotent stem cell (iPSC)
ST:STUDY_SUMMARY                 	and CRISPR technologies offer a powerful tool for this purpose. While typically
ST:STUDY_SUMMARY                 	differentiated into disease-relevant cell types, recent studies support the use
ST:STUDY_SUMMARY                 	of undifferentiated iPSCs for drug discovery. Here, we developed and
ST:STUDY_SUMMARY                 	characterized NDUFS4 KO iPSCs in their pluripotent state. Metabolomic profiling
ST:STUDY_SUMMARY                 	revealed a distinct phenotype in NDUFS4 KO iPSCs, predominantly associated with
ST:STUDY_SUMMARY                 	an elevated NADH/NAD+ ratio, consistent with alterations observed in other
ST:STUDY_SUMMARY                 	models of mitochondrial dysfunction. These findings underscore the potential of
ST:STUDY_SUMMARY                 	iPSCs for early-stage, high-throughput therapeutic screening in mitochondrial
ST:STUDY_SUMMARY                 	diseases.
ST:INSTITUTE                     	North-West University
ST:LAST_NAME                     	Louw
ST:FIRST_NAME                    	Roan
ST:ADDRESS                       	Hoffman street
ST:EMAIL                         	Roan.Louw@nwu.ac.za
ST:PHONE                         	+27182994074
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	-	WT7_1	Genotype:WT | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=WT7_1.d; RAW_FILE_NAME(GCTOF file name)=W7_1.cdf; RAW_FILE_NAME(GC-QQQ file name)=W71_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=W71_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=W71_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	WT7_2	Genotype:WT | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=WT7_2.d; RAW_FILE_NAME(GCTOF file name)=W7_2.cdf; RAW_FILE_NAME(GC-QQQ file name)=W72_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=W72_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=W72_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	WT7_3	Genotype:WT | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=WT7_3.d; RAW_FILE_NAME(GCTOF file name)=W7_3.cdf; RAW_FILE_NAME(GC-QQQ file name)=W73_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=W73_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=W73_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	WT7_4	Genotype:WT | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=WT7_4.d; RAW_FILE_NAME(GCTOF file name)=W7_4.cdf; RAW_FILE_NAME(GC-QQQ file name)=W74_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=W74_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=W74_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	WT7_5	Genotype:WT | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=WT7_5.d; RAW_FILE_NAME(GCTOF file name)=W7_5.cdf; RAW_FILE_NAME(GC-QQQ file name)=W75_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=W75_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=W75_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	KO5_1	Genotype:KO | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=KO5_1.d; RAW_FILE_NAME(GCTOF file name)=KO5_1.cdf; RAW_FILE_NAME(GC-QQQ file name)=K51_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=K51_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=K51_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	KO5_2	Genotype:KO | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=KO5_2.d; RAW_FILE_NAME(GCTOF file name)=KO5_2.cdf; RAW_FILE_NAME(GC-QQQ file name)=K52_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=K52_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=K52_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	KO5_3	Genotype:KO | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=KO5_3.d; RAW_FILE_NAME(GCTOF file name)=KO5_3.cdf; RAW_FILE_NAME(GC-QQQ file name)=K53_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=K53_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=K53_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	KO5_4	Genotype:KO | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=KO5_4.d; RAW_FILE_NAME(GCTOF file name)=KO5_4.cdf; RAW_FILE_NAME(GC-QQQ file name)=K54_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=K54_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=K54_Method3.d
SUBJECT_SAMPLE_FACTORS           	-	KO5_5	Genotype:KO | Sample source:Stem cells	RAW_FILE_NAME(LC file name)=KO5_5.d; RAW_FILE_NAME(GCTOF file name)=KO5_5.cdf; RAW_FILE_NAME(GC-QQQ file name)=K55_Method1.d; RAW_FILE_NAME(GC-QQQ file name)=K55_Method2.d; RAW_FILE_NAME(GC-QQQ file name)=K55_Method3.d
#COLLECTION
CO:COLLECTION_SUMMARY            	Five replicate samples per genotype (i.e. distinct cultures in parallel) were
CO:COLLECTION_SUMMARY            	prepared for each metabolic analysis. Each cell pellet, harvested from a T25
CO:COLLECTION_SUMMARY            	culture flasks, was washed three times with chilled PBS before being quenched in
CO:COLLECTION_SUMMARY            	cold HPLC-grade methanol, with subsequent addition of an internal standard
CO:COLLECTION_SUMMARY            	mixture in cold HPLC-grade water. Thereafter, samples were homogenized using a
CO:COLLECTION_SUMMARY            	vibration mill (30 Hz, 1 min) and incubated on ice (10 min) after the addition
CO:COLLECTION_SUMMARY            	of cold HPLC-grade chloroform. Solvents were added in a ratio of 3:1:1,
CO:COLLECTION_SUMMARY            	methanol/water/chloroform, as required for a modified monophasic Bligh-Dyer
CO:COLLECTION_SUMMARY            	extraction. After centrifugation at 12 000 ×g (10 min, 4°C) supernatants were
CO:COLLECTION_SUMMARY            	aliquoted into 2 mL glass vials.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	N/A
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Samples were derivatized by butylation on the day of analysis. To butylate the
SP:SAMPLEPREP_SUMMARY            	dried extracts, 300 μL of freshly prepared 1-butanol:acetyl chloride (4:1,
SP:SAMPLEPREP_SUMMARY            	v/v) was added and samples were incubated at 50 °C for 60 min. Thereafter,
SP:SAMPLEPREP_SUMMARY            	the samples were evaporated to dryness under a gentle stream of nitrogen at 37
SP:SAMPLEPREP_SUMMARY            	°C. The samples were then reconstituted in 100 μL of water:acetonitrile
SP:SAMPLEPREP_SUMMARY            	(50:50, v/v), containing 0.1% formic acid and vortex mixed. Finally, the total
SP:SAMPLEPREP_SUMMARY            	volume was transferred to 250 μL tapered glass inserts placed in 2 mL vials
SP:SAMPLEPREP_SUMMARY            	and loaded onto the autosampler for analysis. 
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890B
CH:COLUMN_NAME                   	Restek Rxi-1MS (30m x 0.32mm x 0.25μm)
CH:SOLVENT_A                     	N/A
CH:SOLVENT_B                     	N/A
CH:FLOW_GRADIENT                 	N/A
CH:FLOW_RATE                     	1 mL/min
CH:COLUMN_TEMPERATURE            	70 °C for 1 min; followed by a ramp at 12 °C/min until reaching 135 °C, held
CH:COLUMN_TEMPERATURE            	for 1.5 min; a ramp at 2 °C/min until reaching 145 °C, held for 1.5 min; and a
CH:COLUMN_TEMPERATURE            	ramp of 23 °C/min until reaching a final temperature of 300 °C, held for 1 min
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 7010B
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Redox-related organic acids were analysed by multiple reaction monitoring of the
MS:MS_COMMENTS                   	transition from precursor to product ions at associated optimized collision
MS:MS_COMMENTS                   	energies, and fragmentor voltages using Agilent Masshunter. To increase
MS:MS_COMMENTS                   	analytical sensitivity, dMRMs were divided into three methods (identical
MS:MS_COMMENTS                   	chromatography), thus requiring three separate sample injections to analyse all
MS:MS_COMMENTS                   	compounds.To achieve the highest confidence level in metabolite identities, two
MS:MS_COMMENTS                   	unique transitions were monitored per metabolite, thus, allowing spectral and
MS:MS_COMMENTS                   	retention time matching
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Normalised peak areas
MS_METABOLITE_DATA_START
Samples	WT7_1	WT7_2	WT7_3	WT7_4	WT7_5	KO5_1	KO5_2	KO5_3	KO5_4	KO5_5
Factors	Genotype:WT | Sample source:Stem cells	Genotype:WT | Sample source:Stem cells	Genotype:WT | Sample source:Stem cells	Genotype:WT | Sample source:Stem cells	Genotype:WT | Sample source:Stem cells	Genotype:KO | Sample source:Stem cells	Genotype:KO | Sample source:Stem cells	Genotype:KO | Sample source:Stem cells	Genotype:KO | Sample source:Stem cells	Genotype:KO | Sample source:Stem cells
2-Keto-3-methylbutyrate	0.435	0.466	0.368	0.424	0.388	0.343	0.468	0.378	0.39	0.377
2-Keto-3-methylvalerate	0.093	0.108	0.102	0.114	0.108	0.082	0.115	0.103	0.148	0.136
2-Ketoglutarate	0.088	0.071	0.086	0.09	0.086	0.068	0.066	0.069	0.073	0.079
2-Hydroxy-3-methylvalerate	10.433	11.03	11.585	11.231	12.232	14.735	16.109	16.441	17.212	17.714
2-Hydroxy-4-methylvalerate	0.676	0.609	0.799	0.636	0.819	0.525	0.496	0.593	0.578	0.604
2-Hydroxyglutarate	0.008	0.008	0.008	0.006	0.005	0.005	0.004	0.004	0.005	0.005
3-Ketodecanoate	0.156	0.15	0.205	0.192	0.182	0.309	0.164	0.239	0.19	0.189
3-Hydroxybutyrate	0.081	0.085	0.069	0.075	0.078	0.08	0.104	0.098	0.107	0.093
3-Hydroxyisovalerate	0.121	0.115	0.131	0.108	0.13	0.088	0.094	0.124	0.113	0.132
3-Hydroxypalmitate	0.195	0.161	0.194	0.163	0.192	0.178	0.16	0.156	0.159	0.167
Acetoacetate	0.222	0.241	0.198	0.219	0.158	0.258	0.303	0.241	0.24	0.254
Citrate	0.273	0.27	0.294	0.276	0.299	0.268	0.254	0.252	0.258	0.262
Lactate	0.862	0.856	0.87	0.89	0.922	0.904	0.891	0.898	0.888	0.889
Aspartate	0.048	0.056	0.044	0.035	0.025	0.035	0.047	0.043	0.049	0.052
Malate	0.068	0.065	0.065	0.056	0.042	0.044	0.045	0.045	0.047	0.043
Pyruvate	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	PubChem ID
2-Hydroxy-3-methylvalerate	10796774
2-Hydroxy-4-methylvalerate	92779
2-Hydroxyglutarate	43
2-Keto-3-methylbutyrate	49
2-Keto-3-methylvalerate	439286
2-Ketoglutarate	51
3-Hydroxybutyrate	441
3-Hydroxyisovalerate	69362
3-Hydroxypalmitate	10989404
Acetoacetate	6971017
Aspartate	5960
Citrate	311
Lactate	107689
Malate	222656
Pyruvate	1060
3-Ketodecanoate	5282982
METABOLITES_END
#END