#METABOLOMICS WORKBENCH Sethjparker_20241101_174922 DATATRACK_ID:5336 STUDY_ID:ST003558 ANALYSIS_ID:AN005849 PROJECT_ID:PR002192 VERSION 1 CREATED_ON November 4, 2024, 1:42 pm #PROJECT PR:PROJECT_TITLE N-methyl-arginine uptake by HEK293 cells is inhibited by cationic amino acids. PR:PROJECT_TYPE Manuscript PR:PROJECT_SUMMARY N-methyl-arginine is a transported substrate and inhibitor of lysine uptake in PR:PROJECT_SUMMARY cells. In this project, we aimed to determine whether N-methyl-arginine uptake PR:PROJECT_SUMMARY is inhibited by cationic and/or neutral amino acid transporters by co-treating PR:PROJECT_SUMMARY cells with N-methyl-arginine and one of the following amino acids: D-lysine, PR:PROJECT_SUMMARY L-lysine, L-ornithine, L-leucine, L-alanine, or L-arginine. N-methyl-arginine PR:PROJECT_SUMMARY uptake was inhibited by all cationic amino acids and L-leucine but not PR:PROJECT_SUMMARY L-alanine. To determine whether the SLC7 family of transporter(s) are necessary PR:PROJECT_SUMMARY for N-methyl-arginine transport, we also pre-treated cells with the alkylating PR:PROJECT_SUMMARY agent N-ethylmaleimide (NEM) prior to supplementing N-methyl-arginine. SLC7 PR:PROJECT_SUMMARY transporters have a conserved reduced thiol that resides within the binding PR:PROJECT_SUMMARY pocket and is sensitive to NEM alkylation. N-methyl-arginine uptake was not PR:PROJECT_SUMMARY significantly affected by NEM pre-treatment, suggesting that transport is PR:PROJECT_SUMMARY through multiple cationic amino acid transporters. 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 Competitive inhibition of N-methyl-arginine uptake in HEK293 cells treated with ST:STUDY_TITLE cationic and neutral amino acids. ST:STUDY_SUMMARY N-methyl-arginine uptake analysis was performed by supplementing cells with 0.5 ST:STUDY_SUMMARY mM of N-methyl-arginine and a vehicle control (PBS) or 5 mM of one of the ST:STUDY_SUMMARY following amino acids: L-lysine, D-lysine, L-ornithine, L-arginine, L-leucine, ST:STUDY_SUMMARY or L-alanine. In a subset of experiments, the cells were pretreated with 0.2 mM ST:STUDY_SUMMARY of N-ethylmaleimide (NEM) for 15-minutes to alkylate SLC7 transporters. After a ST:STUDY_SUMMARY 1-hour treatment with N-methyl-arginine, cells were quickly washed with ice cold ST:STUDY_SUMMARY saline following by quenching and metabolite extraction using 1 mL of 80% ice ST:STUDY_SUMMARY cold methanol containing 1 ul of stable isotope-labeled internal amino acids ST:STUDY_SUMMARY standards. After vortexing at 4C for 10 minutes and centrifuging, 0.9 ml of ST:STUDY_SUMMARY 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 - DLYS1_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=DLYS1_R1.mzXML SUBJECT_SAMPLE_FACTORS - DLYS1_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=DLYS1_R2.mzXML SUBJECT_SAMPLE_FACTORS - DLYS1_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=DLYS1_R3.mzXML SUBJECT_SAMPLE_FACTORS - DLYS2_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=DLYS2_R1.mzXML SUBJECT_SAMPLE_FACTORS - DLYS2_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=DLYS2_R2.mzXML SUBJECT_SAMPLE_FACTORS - DLYS2_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=DLYS2_R3.mzXML SUBJECT_SAMPLE_FACTORS - LALA1_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LALA1_R1.mzXML SUBJECT_SAMPLE_FACTORS - LALA1_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LALA1_R2.mzXML SUBJECT_SAMPLE_FACTORS - LALA1_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LALA1_R3.mzXML SUBJECT_SAMPLE_FACTORS - LALA2_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LALA2_R1.mzXML SUBJECT_SAMPLE_FACTORS - LALA2_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LALA2_R2.mzXML SUBJECT_SAMPLE_FACTORS - LALA2_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LALA2_R3.mzXML SUBJECT_SAMPLE_FACTORS - LARG1_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LARG1_R1.mzXML SUBJECT_SAMPLE_FACTORS - LARG1_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LARG1_R2.mzXML SUBJECT_SAMPLE_FACTORS - LARG1_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LARG1_R3.mzXML SUBJECT_SAMPLE_FACTORS - LARG2_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LARG2_R1.mzXML SUBJECT_SAMPLE_FACTORS - LARG2_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LARG2_R2.mzXML SUBJECT_SAMPLE_FACTORS - LARG2_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LARG2_R3.mzXML SUBJECT_SAMPLE_FACTORS - LLEU1_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LLEU1_R1.mzXML SUBJECT_SAMPLE_FACTORS - LLEU1_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LLEU1_R2.mzXML SUBJECT_SAMPLE_FACTORS - LLEU1_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LLEU1_R3.mzXML SUBJECT_SAMPLE_FACTORS - LLEU2_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LLEU2_R1.mzXML SUBJECT_SAMPLE_FACTORS - LLEU2_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LLEU2_R2.mzXML SUBJECT_SAMPLE_FACTORS - LLEU2_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LLEU2_R3.mzXML SUBJECT_SAMPLE_FACTORS - LLYS1_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LLYS1_R1.mzXML SUBJECT_SAMPLE_FACTORS - LLYS1_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LLYS1_R2.mzXML SUBJECT_SAMPLE_FACTORS - LLYS1_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LLYS1_R3.mzXML SUBJECT_SAMPLE_FACTORS - LLYS2_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LLYS2_R1.mzXML SUBJECT_SAMPLE_FACTORS - LLYS2_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LLYS2_R2.mzXML SUBJECT_SAMPLE_FACTORS - LLYS2_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LLYS2_R3.mzXML SUBJECT_SAMPLE_FACTORS - LORN1_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LORN1_R1.mzXML SUBJECT_SAMPLE_FACTORS - LORN1_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LORN1_R2.mzXML SUBJECT_SAMPLE_FACTORS - LORN1_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LORN1_R3.mzXML SUBJECT_SAMPLE_FACTORS - LORN2_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=LORN2_R1.mzXML SUBJECT_SAMPLE_FACTORS - LORN2_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=LORN2_R2.mzXML SUBJECT_SAMPLE_FACTORS - LORN2_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=LORN2_R3.mzXML SUBJECT_SAMPLE_FACTORS - NEMNMA1_R1 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=NEMNMA1_R1.mzXML SUBJECT_SAMPLE_FACTORS - NEMNMA1_R2 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=NEMNMA1_R2.mzXML SUBJECT_SAMPLE_FACTORS - NEMNMA1_R3 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=NEMNMA1_R3.mzXML SUBJECT_SAMPLE_FACTORS - NEMNMA2_R1 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=NEMNMA2_R1.mzXML SUBJECT_SAMPLE_FACTORS - NEMNMA2_R2 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=NEMNMA2_R2.mzXML SUBJECT_SAMPLE_FACTORS - NEMNMA2_R3 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=NEMNMA2_R3.mzXML SUBJECT_SAMPLE_FACTORS - NEMPBS1_R1 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=NEMPBS1_R1.mzXML SUBJECT_SAMPLE_FACTORS - NEMPBS1_R2 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=NEMPBS1_R2.mzXML SUBJECT_SAMPLE_FACTORS - NEMPBS1_R3 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=NEMPBS1_R3.mzXML SUBJECT_SAMPLE_FACTORS - NEMPBS2_R1 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=NEMPBS2_R1.mzXML SUBJECT_SAMPLE_FACTORS - NEMPBS2_R2 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=NEMPBS2_R2.mzXML SUBJECT_SAMPLE_FACTORS - NEMPBS2_R3 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=NEMPBS2_R3.mzXML SUBJECT_SAMPLE_FACTORS - NMA1_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=NMA1_R1.mzXML SUBJECT_SAMPLE_FACTORS - NMA1_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=NMA1_R2.mzXML SUBJECT_SAMPLE_FACTORS - NMA1_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=NMA1_R3.mzXML SUBJECT_SAMPLE_FACTORS - NMA2_R1 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=NMA2_R1.mzXML SUBJECT_SAMPLE_FACTORS - NMA2_R2 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=NMA2_R2.mzXML SUBJECT_SAMPLE_FACTORS - NMA2_R3 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=NMA2_R3.mzXML SUBJECT_SAMPLE_FACTORS - PBS1_R1 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=PBS1_R1.mzXML SUBJECT_SAMPLE_FACTORS - PBS1_R2 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=PBS1_R2.mzXML SUBJECT_SAMPLE_FACTORS - PBS1_R3 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=PBS1_R3.mzXML SUBJECT_SAMPLE_FACTORS - PBS2_R1 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=1; RAW_FILE_NAME(Raw file name)=PBS2_R1.mzXML SUBJECT_SAMPLE_FACTORS - PBS2_R2 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=2; RAW_FILE_NAME(Raw file name)=PBS2_R2.mzXML SUBJECT_SAMPLE_FACTORS - PBS2_R3 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Independent experiment=3; RAW_FILE_NAME(Raw file name)=PBS2_R3.mzXML #COLLECTION CO:COLLECTION_SUMMARY Cells were briefly washed with ice cold saline before adding 1 mL of ice cold CO:COLLECTION_SUMMARY 80% methanol to quench metabolism. Cell extracts were scraped using a p1000 tip, CO:COLLECTION_SUMMARY transferred to a tube, vortexed at 4C for 10 minutes, centrifuged at 21,000xg CO:COLLECTION_SUMMARY for 15 minutes, and dried for LCMS analysis. CO:SAMPLE_TYPE Epithelial cells #TREATMENT TR:TREATMENT_SUMMARY Cells were plated in DMEM containing 10% FBS and allowed to attach overnight. TR:TREATMENT_SUMMARY The next morning, the media was aspirated and cells were pretreated with 0.2 mM TR:TREATMENT_SUMMARY NEM for 15 minutes or immediately supplemented with 5 mM of L-lysine, D-lysine, TR:TREATMENT_SUMMARY L-ornithine, L-arginine, L-leucine, or L-alanine and/or 0.5 mM N-methyl-arginine TR:TREATMENT_SUMMARY for 1-hour. All uptake experiments were performed in PBS. TR:TREATMENT_COMPOUND N-methyl-arginine and other amino acids TR:TREATMENT_DOSE 0.5 mM for NMA and 5 mM for indicated amino acids 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/1e6 cells MS_METABOLITE_DATA_START Samples PBS1_R1 PBS2_R1 NMA1_R1 NMA2_R1 NEMPBS1_R1 NEMPBS2_R1 NEMNMA1_R1 NEMNMA2_R1 LLYS1_R1 LLYS2_R1 DLYS1_R1 DLYS2_R1 LARG1_R1 LARG2_R1 LORN1_R1 LORN2_R1 LALA1_R1 LALA2_R1 LLEU1_R1 LLEU2_R1 PBS1_R2 PBS2_R2 NMA1_R2 NMA2_R2 NEMPBS1_R2 NEMPBS2_R2 NEMNMA1_R2 NEMNMA2_R2 LLYS1_R2 LLYS2_R2 DLYS1_R2 DLYS2_R2 LARG1_R2 LARG2_R2 LORN1_R2 LORN2_R2 LALA1_R2 LALA2_R2 LLEU1_R2 LLEU2_R2 PBS1_R3 PBS2_R3 NMA1_R3 NMA2_R3 NEMPBS1_R3 NEMPBS2_R3 NEMNMA1_R3 NEMNMA2_R3 LLYS1_R3 LLYS2_R3 DLYS1_R3 DLYS2_R3 LARG1_R3 LARG2_R3 LORN1_R3 LORN2_R3 LALA1_R3 LALA2_R3 LLEU1_R3 LLEU2_R3 Factors Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:PBS | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:N-ethylmaleimide (0.2 mM) | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:None | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:D-lysine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-arginine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-ornithine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-alanine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 Pre-treatment:None | Treatment 1:N-methyl-arginine (0.5 mM) | Treatment 2:L-leucine (5 mM) | Sample source:HEK293 NG-METHYL-ARGININE 895268.9357 1431434.24 2762507722 2903107456 3509546.468 3680486.442 4083974559 4256128698 1157456555 1060733667 2018979060 1911976901 678543931.1 639405035.9 747625120.3 828720835.2 2492202972 2534410474 1448675775 1501009194 1404980.736 2381106.474 3542542873 3456049874 1339255.899 2102984.139 2954850557 2807582467 1616481116 1375724050 2197385045 2183338645 1101837063 997559425.8 1009028907 1098651894 7741832601 7036834623 7563981383 6961756255 2071160.369 1767415.381 3123855509 3176454456 6703462.074 6536304.462 3812260826 3959301452 1469098348 1282834587 2042482427 2074894985 1262308682 1087765526 1023730988 1193837149 2752917907 2655670917 1569114030 1618480666 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Chemical Formula RT Mass (m/z) Category NG-METHYL-ARGININE C7H16N4O2 26.1 189.1346 HMDB0000182 METABOLITES_END #END