#METABOLOMICS WORKBENCH Karin_20241006_114144 DATATRACK_ID:5264 STUDY_ID:ST003544 ANALYSIS_ID:AN005822 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 8890 CH:COLUMN_NAME Restek Rxi-5MS (29.69m x 0.25mm,0.25um) 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 10 °C/min until reaching a final CH:COLUMN_TEMPERATURE temperature of 320 °C, held for 3 min #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Leco Pegasus BT TOF MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass spectra (50–850 m/z) were acquired at a scan rate of 20 spectra/s after a MS:MS_COMMENTS 300-s solvent delay.The ChromaTOF software (LECO Corp., version 4.51) was used MS:MS_COMMENTS for data acquisition, processing, and extraction. Peaks with similar mass MS:MS_COMMENTS spectra and retention times were aligned using the Statistical Compare package. MS:MS_RESULTS_FILE ST003544_AN005822_Results.txt UNITS:Normalised peak areas Has m/z:Yes Has RT:Yes RT units:Seconds #END