#METABOLOMICS WORKBENCH Karin_20241006_114144 DATATRACK_ID:5264 STUDY_ID:ST003544 ANALYSIS_ID:AN005820 PROJECT_ID:PR002180
VERSION                          	1
CREATED_ON                       	03-03-2025
#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
PR:DOI                           	http://dx.doi.org/10.21228/M8TV67
#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                       	Hofman Street, Potchefstroom, North-West, 2520, South Africa
ST:EMAIL                         	Roan.Louw@nwu.ac.za
ST:PHONE                         	+27182994074
ST:SUBMIT_DATE                   	2024-10-06
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:SPECIES_GROUP                 	Mammals
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
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
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
#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, the
SP:SAMPLEPREP_SUMMARY            	samples were evaporated to dryness under a gentle stream of nitrogen at 37 °C.
SP:SAMPLEPREP_SUMMARY            	The samples were then reconstituted in 100 μL of water:acetonitrile (50:50,
SP:SAMPLEPREP_SUMMARY            	v/v), containing 0.1% formic acid and vortex mixed. Finally, the total volume
SP:SAMPLEPREP_SUMMARY            	was transferred to 250 μL tapered glass inserts placed in 2 mL vials and
SP:SAMPLEPREP_SUMMARY            	loaded onto the autosampler for analysis. 
#CHROMATOGRAPHY
CH:INSTRUMENT_NAME               	Agilent 1200 Infinity
CH:COLUMN_NAME                   	Agilent C18 Zorbax SB-AQ (100 x 2.1mm, 1.8um)
CH:COLUMN_TEMPERATURE            	45°C
CH:FLOW_GRADIENT                 	95 % of solvent A held for 0.2 min, increased to 25 % of solvent B over 1.8 min,
CH:FLOW_GRADIENT                 	held for 5 min, increased to 90 % of solvent B over 0.5 min, held for 1.6 min,
CH:FLOW_GRADIENT                 	increased to 95 % of solvent B over 2.9 min, decreased to 5 % of solvent B over
CH:FLOW_GRADIENT                 	1 min, and re-equilibrated for 3 min to give a total run time of 16 min.
CH:FLOW_RATE                     	For the first 9 min of the run, the mobile phase flow rate was set at 0.3
CH:FLOW_RATE                     	mL/min, after which it was increased to 0.4 mL/min in a span of 0.1 min and
CH:FLOW_RATE                     	maintained at this level for the subsequent 3.9 min of the run
CH:SOLVENT_A                     	100% Water; 0.1 % Formic acid
CH:SOLVENT_B                     	100% Acetonitrile; 0.1 % Formic acid
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6410 QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	Amino acid and acylcarnitine species were analyzed by dynamic multiple reaction
MS:MS_COMMENTS                   	monitoring of the transition from precursor to product ions at associated
MS:MS_COMMENTS                   	optimized collision energies, and fragmentor voltages using Agilent Masshunter.
MS:MS_COMMENTS                   	To achieve the highest confidence level in metabolite identities, two unique
MS:MS_COMMENTS                   	transitions were monitored per metabolite, thus, allowing spectral and retention
MS:MS_COMMENTS                   	time matching
MS:ION_MODE                      	POSITIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	Normalized peak area
MS_METABOLITE_DATA_START
Samples	KO5_1	KO5_2	KO5_3	KO5_4	KO5_5	WT7_1	WT7_2	WT7_3	WT7_4	WT7_5
Factors	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	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	
1-Methylhistidine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
2-Aminoadipic acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
3-Aminoisobutanoic acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
4-Hydroxyproline	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Beta-Alanine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Betaine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Citrulline	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Creatine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Cystine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Decanoylcarnitine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
gamma-Aminobutyric acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Glutamic acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Glutamine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Glycine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Histidine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Isoleucine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Kynurenine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Acetylcarnitine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Alanine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Arginine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Asparagine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Aspartic acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Carnitine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Cystathionine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Leucine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Threonine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Tryptophan	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Tyrosine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
L-Valine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Lysine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Methionine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
N-Acetyl-L-aspartic acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
N-Acetyl-L-glutamic acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Octanoylcarnitine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Ornithine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Palmitoylcarnitine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Phenylalanine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Pipecolic acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Proline	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Pyroglutamic acid	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Serine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
Stearoylcarnitine	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	pubchem_id	inchi_key	kegg_id	other_id	other_id_type	ri	ri_type	moverz_quant	
1-Methylhistidine	92105								
2-Aminoadipic acid	469								
3-Aminoisobutanoic acid	64956								
4-Hydroxyproline	5810								
Beta-Alanine	239								
Betaine	247								
Citrulline	9750								
Creatine	586								
Cystine	595								
Decanoylcarnitine	11953821								
gamma-Aminobutyric acid	119								
Glutamic acid	33032								
Glutamine	5961								
Glycine	750								
Histidine	6274								
Isoleucine	6306								
Kynurenine	161166								
L-Acetylcarnitine	7045767								
L-Alanine	5950								
L-Arginine	6322								
L-Asparagine	6267								
L-Aspartic acid	5960								
L-Carnitine	10917								
L-Cystathionine	439258								
Leucine	6106								
L-Threonine	6288								
L-Tryptophan	6305								
L-Tyrosine	6057								
L-Valine	6287								
Lysine	5962								
Methionine	6137								
N-Acetyl-L-aspartic acid	65065								
N-Acetyl-L-glutamic acid	70914								
Octanoylcarnitine	11953814								
Ornithine	6262								
Palmitoylcarnitine	11953816								
Phenylalanine	6140								
Pipecolic acid	849								
Proline	145742								
Pyroglutamic acid	7405								
Serine	5951								
Stearoylcarnitine	52922056								
METABOLITES_END
#END