#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