#METABOLOMICS WORKBENCH Sethjparker_20241101_175048 DATATRACK_ID:5337 STUDY_ID:ST003559 ANALYSIS_ID:AN005850 PROJECT_ID:PR002193 VERSION 1 CREATED_ON November 5, 2024, 9:00 am #PROJECT PR:PROJECT_TITLE Knockdown of SLC7A1, SLC7A2, or SLC7A6 affects cationic amino acid levels but PR:PROJECT_TITLE not N-methyl-arginine uptake in HEK293 cells. PR:PROJECT_TYPE Manuscript PR:PROJECT_SUMMARY N-methyl-arginine is a cationic amino acid transporter inhibitor that restricts PR:PROJECT_SUMMARY the uptake and metabolism of lysine. Towards understanding how specific cationic PR:PROJECT_SUMMARY amino acid transporters influence lysine and arginine uptake, we performed PR:PROJECT_SUMMARY knockdown studies targeting SLC7A1 (CAT1), SLC7A2 (CAT2), or SLC7A6 (y+LAT2) and PR:PROJECT_SUMMARY performed metabolomics and N-methyl-arginine uptake assays. Our results suggest PR:PROJECT_SUMMARY that CAT1 and CAT2 facilitate the import of lysine, whereas y+LAT2 facilitates PR:PROJECT_SUMMARY cellular secretion in exchange for a neutral amino acid. N-methyl-arginine PR:PROJECT_SUMMARY uptake was not significantly affected by knockdown of any single transporter, PR:PROJECT_SUMMARY suggesting that it is taken up by multiple cationic amino acid transporters PR:PROJECT_SUMMARY simultaneously. PR:INSTITUTE University of British Columbia PR:DEPARTMENT Biochemistry & Molecular Biology PR:LABORATORY Parker laboratory PR:LAST_NAME Parker PR:FIRST_NAME Seth PR:ADDRESS 950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada PR:EMAIL seth.parker@bcchr.ca PR:PHONE 6048753121 #STUDY ST:STUDY_TITLE Metabolomics and N-methyl-arginine uptake in control (siNTC) and cationic amino ST:STUDY_TITLE acid transporter knockdown HEK293 cells. ST:STUDY_SUMMARY Metabolite analysis of HEK293 cells transfected with a non-targeting siRNA ST:STUDY_SUMMARY (siNTC) or a siRNA targeting either SLC7A1 (CAT1), SLC7A2 (CAT2), or SLC7A6 ST:STUDY_SUMMARY (y+LAT2). Analysis was performed four days after transfection, and knockdown was ST:STUDY_SUMMARY confirmed using qPCR for each experiment. Each independent experiment was ST:STUDY_SUMMARY performed on an independent batch of cells transfected with siRNAs. For ST:STUDY_SUMMARY metabolomics, cells were plated at day 2, and the media was changed to DMEM ST:STUDY_SUMMARY containing 10% dialyzed FBS at day 3. For NMA uptake assays, cells were plated ST:STUDY_SUMMARY at day 2 and NMA uptake was assessed by supplementing cells with PBS or PBS ST:STUDY_SUMMARY containing 0.5 mM NMA for 1 hour at day 4. All samples were processed by ST:STUDY_SUMMARY extracting metabolites using 1 mL of ice cold 80% methanol containing internal, ST:STUDY_SUMMARY isotope-labeled amino acid standards. Metabolites were concentrated using a ST:STUDY_SUMMARY SpeedVac until dry. For plasma metabolite analysis, 10 µl of plasma was added ST:STUDY_SUMMARY to 250 µl of 80% methanol containing 1 µl of stable isotope-labeled internal ST:STUDY_SUMMARY amino acid standard mix, vortexed at 4C for 10 minutes, and centrifuged. 0.9 ml ST:STUDY_SUMMARY of supernatant was transferred to a new tube and concentrated using a SpeedVac ST:STUDY_SUMMARY until dry. Metabolites were reconstituted into 25-50 µl of water, vortexed, ST:STUDY_SUMMARY centrifuged, and transferred to vials for analysis by LCMS. LCMS was performed ST:STUDY_SUMMARY using a ZIC-pHILIC LC column coupled to a Vanquish LC and a flow gradient ST:STUDY_SUMMARY consisting of 10 mM ammonium carbonate in water and pure acetonitrile. The LC ST:STUDY_SUMMARY was coupled to an Exploris 240 mass spectrometer operated in a polarity ST:STUDY_SUMMARY switching data-dependent Top 5 mode. Full MS scan parameters for both positive ST:STUDY_SUMMARY and negative mode were set to 67-1000 m/z at a resolution of 120k and ddMS2 were ST:STUDY_SUMMARY collected at a resolution of 30k. ST:INSTITUTE University of British Columbia ST:DEPARTMENT Biochemistry & Molecular Biology ST:LABORATORY Parker laboratory ST:LAST_NAME Parker ST:FIRST_NAME Seth ST:ADDRESS 950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada ST:EMAIL seth.parker@bcchr.ca ST:PHONE 6048753121 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Not applicable #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 - HEK293_siNTC_d4_PM1 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=1; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_d4_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_d4_PM2 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=1; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_d4_PM2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_d4_PM1 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Experiment=Metabolomics; Independent experiment=1; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_d4_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_d4_PM2 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Experiment=Metabolomics; Independent experiment=1; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_d4_PM2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_PM3 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=2; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_PM3.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_PM4 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Experiment=Metabolomics; Independent experiment=2; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_PM4.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_PM3 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Experiment=Metabolomics; Independent experiment=2; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_PM3.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_PM4 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=2; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_PM4.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_PM6 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Experiment=Metabolomics; Independent experiment=3; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_PM6.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_PM6 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=3; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_PM6.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_PM5 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Experiment=Metabolomics; Independent experiment=3; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_PM5.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_PM5 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=3; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_PM5.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_PM1 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT2_PM1 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Experiment=Metabolomics; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siCAT2_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_PM2 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_PM2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT2_PM2 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Experiment=Metabolomics; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siCAT2_PM2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_PM2 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Experiment=Metabolomics; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_PM2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_PM1 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Experiment=Metabolomics; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT1_PM1 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Experiment=Metabolomics; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siCAT1_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT1_PM2 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Experiment=Metabolomics; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siCAT1_PM2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT2_PM4 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Experiment=Metabolomics; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_siCAT2_PM4.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT2_PM3 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Experiment=Metabolomics; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_siCAT2_PM3.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT1_PM4 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Experiment=Metabolomics; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_siCAT1_PM4.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT1_PM3 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Experiment=Metabolomics; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_siCAT1_PM3.RAW SUBJECT_SAMPLE_FACTORS - HEK293_NTC_r1_0821 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_NTC_r1_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT2_r1_0821 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Experiment=Metabolomics; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_CAT2_r1_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_NTC_r2_0821 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Experiment=Metabolomics; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_NTC_r2_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT2_r2_0821 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Experiment=Metabolomics; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_CAT2_r2_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT1_r1_0821 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Experiment=Metabolomics; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_CAT1_r1_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT1_r2_0821 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Experiment=Metabolomics; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_CAT1_r2_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_PBS_PM1 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Experiment=NMA uptake; Independent experiment=1; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_PBS_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_NMA_PM1 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=1; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_NMA_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_PBS_PM1 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Experiment=NMA uptake; Independent experiment=1; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_PBS_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_NMA_PM1 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=1; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_NMA_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_NMA_r2 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=2; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_NMA_r2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_noNMA_r2 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Experiment=NMA uptake; Independent experiment=2; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_noNMA_r2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_NMA_r2 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=2; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_NMA_r2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_noNMA_r2 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Experiment=NMA uptake; Independent experiment=2; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_noNMA_r2.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_noNMA_3 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Experiment=NMA uptake; Independent experiment=3; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_noNMA_3.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_noNMA_3 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Experiment=NMA uptake; Independent experiment=3; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_noNMA_3.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_NMA_3 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=3; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_NMA_3.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siSLC7A6_NMA_3 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=3; RAW_FILE_NAME(Raw file name)=HEK293_siSLC7A6_NMA_3.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_wNMA_PM1 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_wNMA_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT1_wNMA_PM1 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siCAT1_wNMA_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siNTC_noNMA_PM1 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Experiment=NMA uptake; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siNTC_noNMA_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT2_noNMA_PM1 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:PBS Experiment=NMA uptake; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siCAT2_noNMA_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT1_noNMA_PM1 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:PBS Experiment=NMA uptake; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siCAT1_noNMA_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_siCAT2_wNMA_PM1 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=4; RAW_FILE_NAME(Raw file name)=HEK293_siCAT2_wNMA_PM1.RAW SUBJECT_SAMPLE_FACTORS - HEK293_NTC_NMA_0821 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_NTC_NMA_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_7A6_NMA_0821 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_7A6_NMA_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_7A6_noNMA_0821 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Experiment=NMA uptake; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_7A6_noNMA_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT1_noNMA_0821 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:PBS Experiment=NMA uptake; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_CAT1_noNMA_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT2_noNMA_0821 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:PBS Experiment=NMA uptake; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_CAT2_noNMA_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT2_NMA_0821 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_CAT2_NMA_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_NTC_noNMA_0821 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Experiment=NMA uptake; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_NTC_noNMA_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT1_NMA_0821 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=5; RAW_FILE_NAME(Raw file name)=HEK293_CAT1_NMA_0821.RAW SUBJECT_SAMPLE_FACTORS - HEK293_7A6_noNMA_0823 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Experiment=NMA uptake; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_7A6_noNMA_0823.RAW SUBJECT_SAMPLE_FACTORS - HEK293_NTC_noNMA_0823 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Experiment=NMA uptake; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_NTC_noNMA_0823.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT1_NMA_0823 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_CAT1_NMA_0823.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT1_noNMA_0823 Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:PBS Experiment=NMA uptake; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_CAT1_noNMA_0823.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT2_noNMA_0823 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:PBS Experiment=NMA uptake; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_CAT2_noNMA_0823.RAW SUBJECT_SAMPLE_FACTORS - HEK293_CAT2_NMA_0823 Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_CAT2_NMA_0823.RAW SUBJECT_SAMPLE_FACTORS - HEK293_NTC_NMA_0823 Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_NTC_NMA_0823.RAW SUBJECT_SAMPLE_FACTORS - HEK293_7A6_NMA_0823 Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Experiment=NMA uptake; Independent experiment=6; RAW_FILE_NAME(Raw file name)=HEK293_7A6_NMA_0823.RAW #COLLECTION CO:COLLECTION_SUMMARY Cells were briefly washed with ice cold saline prior to metabolite extraction. 1 CO:COLLECTION_SUMMARY mL of ice cold 80% methanol containing internal standards was added to each CO:COLLECTION_SUMMARY well, and cell extracts were collected by scraping wells with a p1000 tip. CO:COLLECTION_SUMMARY Samples were vortexed at 1500 rpm at 4C for 15 minutes and cleared by CO:COLLECTION_SUMMARY centrifugation at 21,000xg for 15 minutes. 900 ul of each sample was transferred CO:COLLECTION_SUMMARY to a new tube and dried using a SpeedVac for analysis by LCMS. CO:SAMPLE_TYPE Epithelial cells #TREATMENT TR:TREATMENT_SUMMARY For metabolomics experiments, no treatment was performed. For N-methyl-arginine TR:TREATMENT_SUMMARY uptake assays, the media was aspirated and cells were treated with PBS or PBS TR:TREATMENT_SUMMARY containing 0.5 mM N-methyl-arginine and incubated for 1-hours. After 1-hour, TR:TREATMENT_SUMMARY cells were washed and processed for metabolite analysis using LCMS. TR:TREATMENT_COMPOUND N-methyl-arginine TR:TREATMENT_DOSE 0.5 mM TR:TREATMENT_VEHICLE PBS #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Dried samples were reconstituted in 50 µL of HPLC-grade water. Samples were SP:SAMPLEPREP_SUMMARY vortexed for ~10 minutes, then centrifuged at 21,000 x g for 15 min at 4°C. 40 SP:SAMPLEPREP_SUMMARY microliters were transferred to LC vials containing glass inserts for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A 100% water, 10 mM Ammonium Carbonate, pH 9.0 CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 80-20%B (0-30 min), 20-20%B (30-40 minute), and 20-80%B (40-40.5 minute); the LC CH:FLOW_GRADIENT column was re-equilibrated using 80-80%B from 40.5-52 minute before subsequent CH:FLOW_GRADIENT injections CH:FLOW_RATE 100 uL/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The LC was coupled to a Thermo Scientific Exploris 240 mass spectrometer MS:MS_COMMENTS operating in heated electrospray ionization mode (HESI) for analysis. The MS:MS_COMMENTS following parameters were set for HESI: spray voltage 3.4 kV (positive) and 2 kV MS:MS_COMMENTS (negative), static spray voltage, sheath gas 25, aux gas 5, sweep gas 0.5, ion MS:MS_COMMENTS transfer tube temperature 320oC, and vaporizer temperature 75oC. The global MS:MS_COMMENTS parameters included an expected peak width of 20 seconds, mild trapping, and a MS:MS_COMMENTS default charge state of 1. A 40-min polarity switching data-dependent Top 5 MS:MS_COMMENTS method was used for positive mode and a data-dependent Top 3 method was used for MS:MS_COMMENTS negative mode. Full MS scan parameters for both positive and negative modes were MS:MS_COMMENTS set as follows: scan range 67-1000 m/z collected in profile mode, Orbitrap MS:MS_COMMENTS resolution 120,000, RF lens 70%, AGC target of 300%, and maximum injection time MS:MS_COMMENTS set to automatic. ddMS2 for positive mode were collected in centroid mode at an MS:MS_COMMENTS Orbitrap resolution of 30,000, isolation window of 1.5 m/z, an AGC target set to MS:MS_COMMENTS standard, a maximum injection time set to automatic, and a normalized collision MS:MS_COMMENTS energy set to 10%, 30%, and 80%. ddMS2 for negative mode were collected in MS:MS_COMMENTS centroid mode at an Orbitrap resolution of 30,000, isolation window of 2 m/z, an MS:MS_COMMENTS AGC target set to standard, a maximum injection time set to automatic, and a MS:MS_COMMENTS normalized collision energy set to 30%. For both positive and negative ddMS2, we MS:MS_COMMENTS applied an intensity threshold of 5e4 and a dynamic exclusion of 5 ppm for 10 MS:MS_COMMENTS seconds, excluding isotopes. A targeted selected ion monitoring (tSIM) scan was MS:MS_COMMENTS also included for pipecolate and P6C/P2C, and the retention time ranges were MS:MS_COMMENTS based on elution of authentic standards (pipecolate) or from positive samples MS:MS_COMMENTS (Aldh7a1-deficient tissues). The tSIM scan for pipecolate was collected from MS:MS_COMMENTS 8-12 minutes in negative mode at an isolation window of 4 m/z (for metabolomics) MS:MS_COMMENTS or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap MS:MS_COMMENTS resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, MS:MS_COMMENTS and collected in profile mode. The tSIM scan for P6C/P2C was collected from 6-10 MS:MS_COMMENTS minutes in positive mode at an isolation window of 4 m/z (for metabolomics) or MS:MS_COMMENTS 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap MS:MS_COMMENTS resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, MS:MS_COMMENTS and collected in profile mode. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion counts MS_METABOLITE_DATA_START Samples HEK293_siNTC_PBS_PM1 HEK293_siNTC_NMA_PM1 HEK293_siSLC7A6_PBS_PM1 HEK293_siSLC7A6_NMA_PM1 HEK293_siNTC_noNMA_r2 HEK293_siNTC_NMA_r2 HEK293_siSLC7A6_noNMA_r2 HEK293_siSLC7A6_NMA_r2 HEK293_siNTC_noNMA_3 HEK293_siNTC_NMA_3 HEK293_siSLC7A6_noNMA_3 HEK293_siSLC7A6_NMA_3 HEK293_siNTC_noNMA_PM1 HEK293_siNTC_wNMA_PM1 HEK293_siCAT1_noNMA_PM1 HEK293_siCAT1_wNMA_PM1 HEK293_siCAT2_noNMA_PM1 HEK293_siCAT2_wNMA_PM1 HEK293_NTC_noNMA_0821 HEK293_NTC_NMA_0821 HEK293_7A6_noNMA_0821 HEK293_7A6_NMA_0821 HEK293_CAT1_noNMA_0821 HEK293_CAT1_NMA_0821 HEK293_CAT2_noNMA_0821 HEK293_CAT2_NMA_0821 HEK293_NTC_noNMA_0823 HEK293_NTC_NMA_0823 HEK293_7A6_noNMA_0823 HEK293_7A6_NMA_0823 HEK293_CAT1_noNMA_0823 HEK293_CAT1_NMA_0823 HEK293_CAT2_noNMA_0823 HEK293_CAT2_NMA_0823 HEK293_siNTC_PM1 HEK293_siNTC_PM2 HEK293_siCAT1_PM1 HEK293_siCAT1_PM2 HEK293_siCAT2_PM1 HEK293_siCAT2_PM2 HEK293_siCAT1_PM3 HEK293_siCAT1_PM4 HEK293_siCAT2_PM3 HEK293_siCAT2_PM4 HEK293_NTC_r1_0821 HEK293_NTC_r2_0821 HEK293_CAT1_r1_0821 HEK293_CAT1_r2_0821 HEK293_CAT2_r1_0821 HEK293_CAT2_r2_0821 HEK293_siNTC_d4_PM1 HEK293_siNTC_d4_PM2 HEK293_siSLC7A6_d4_PM1 HEK293_siSLC7A6_d4_PM2 HEK293_siSLC7A6_PM1 HEK293_siSLC7A6_PM2 HEK293_siNTC_PM3 HEK293_siNTC_PM4 HEK293_siSLC7A6_PM3 HEK293_siSLC7A6_PM4 HEK293_siSLC7A6_PM5 HEK293_siSLC7A6_PM6 HEK293_siNTC_PM5 HEK293_siNTC_PM6 Factors Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:PBS Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:PBS Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:N-methyl-arginine (0.5 mM) Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A1 (CAT1) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A2 (CAT2) | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Sample source:HEK293 | siRNA:siSLC7A6 | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None Sample source:HEK293 | siRNA:siNon-targeting control (siNTC) | Treatment:None LYSINE 1.46E+07 4.64E+06 3.11E+07 5.81E+06 47372410.74 7144134.091 63192058.42 10557136.95 23635044.01 4016142.642 64804374.3 9590590.667 24836880.14 4695898.8 26814306.3 4869682.147 18040307.13 4865448.877 19359192.65 4070429.694 64433060.7 7279739.008 16746019.6 4118084.61 14189685.64 3937037.99 17198735.38 3835180.175 68840387.35 7024534.605 20120085.06 4744658.677 13381769.09 3770617.157 125628741 123180451.8 93924684.83 102562321.7 65837776.51 71141861.97 192473965.9 191297135.9 86182277.57 82636503.75 204978781 201298236.5 138727523.3 129141832.9 83504227.98 86742511.11 218815389.4 221676682.3 276699294.1 280301487.8 183730014.5 178963254.9 153568893.2 166135938.8 176679080.8 190074721 102659609.2 111681881.4 173738293.1 174340894.6 iso_Lys 7.64E+07 6.79E+07 8.23E+07 7.12E+07 92476522.22 75566489.44 89551596.9 74855570.81 78456660.09 68361337.69 81917738.11 67494335.47 93893660.98 77516793.28 94781207.58 77268081.44 88495090.53 80399448.11 91957374.03 79104028.82 97012367.24 76080864.82 91660106.08 77403638.83 94004807.99 82884952.95 92170533.12 76865857.9 89225293.03 73512172.86 88867495.62 74121418.8 87517885.68 78026898.91 37058794.79 36580762.29 38124960.86 38040261.91 34326871.83 34790740.15 34229751.18 33945206.99 32580417.36 32254389.91 34147072.78 33712834.5 34251325.74 32407073.25 32244975.86 34077594.8 82575713.14 83773717.67 81381790.77 81592433.19 37528550.56 38033080.91 76045605.62 80172353.43 80684191.03 85761842.94 76963196.37 78792311.88 76322275.32 76614828.15 ARGININE 1.41E+07 1.76E+07 1.94E+07 1.75E+07 89292311.12 16962904.13 115939409.4 22395187.44 44391015.21 11200768.55 111573798.3 14768839.94 27893691.69 14099945.1 30274958.41 11558498.38 31940896.17 18702565.95 21573316.85 13622057.53 66932764.89 14455866.15 21972233.12 13479177.77 20028246.02 13811655.52 15802078.5 12834258.3 53844630.47 14292135.33 16932492.35 12967024.42 20071306.44 10642018.53 375339463.4 372618202.9 285990686.3 311930365.7 223475584.7 240245451.6 477748545.2 473460987.7 279173087.9 268664486.4 526388164.8 517418037.9 379773961.1 352096189.4 267133334.4 275490727.8 561582916.4 557514588.9 677429683.2 682888212.1 508260563.3 487751734.6 428080161.7 461165841.8 486574856.5 525905051.3 313787726.5 340350772.5 489603380.2 494592455.6 iso_Arg 6.91E+08 6.54E+08 7.02E+08 6.76E+08 720235157.3 688704157.4 707654715.7 661628627.4 689335086.3 672329741.8 701990536.7 657345926 776539185.4 759873040.2 791731828.9 752134905.6 768167382.8 789504266.9 792678786.7 830291503 815718158.5 777518064.3 809927261.6 768906675.8 811729914 818189353.7 799468386 803312972.6 780041473.9 781596278.1 796910900.1 773532788.3 799376235.3 794380295.9 294627665.5 296891884.4 319904983.3 317639225.3 300225322.1 299451096.9 306633379.8 303818886.4 317852917 315494084.4 312362609.4 308347451.1 331492840.9 308318383.5 328553019.7 342459513.6 592134822.4 602807133.5 581279851.7 583607322.3 286482248 286395122.6 568812154.1 600752468.4 600740593.4 639561396 612539690 619851195.1 577221495.9 586306498.5 NG-METHYLARGININE 2.11E+06 8.05E+09 7.08E+06 6.75E+09 7391841.003 5068248352 8301524.314 3817715203 2870855.083 3747431995 3437742.228 3932695615 11342874.21 6875943160 9246823.384 5716386096 6467092.456 4356597166 4457790.369 5360900851 8684673.245 4714517819 337460886.9 4178834878 5660116.721 3282054362 3633878.152 5778170096 5403623.946 5258523884 4559377.871 4507348001 4411717.131 2592165230 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Chemical Formula RT Mass (m/z) Category NG-METHYLARGININE C7H16N4O2 26.1 189.1346 HMDB0000182 iso_Arg C6H14N4O2 27.83 185.12675 standard2 ARGININE C6H14N4O2 27.85 175.119 HMDB0000517 LYSINE C6H14N2O2 26.05 147.11335 HMDB0000182 iso_Lys C6H14N2O2 26.16 155.12656 standard12 METABOLITES_END #END