#METABOLOMICS WORKBENCH akhan01_20250720_120652 DATATRACK_ID:6199 STUDY_ID:ST004060 ANALYSIS_ID:AN006719 PROJECT_ID:PR002544 VERSION 1 CREATED_ON July 21, 2025, 2:57 pm #PROJECT PR:PROJECT_TITLE Machine learning identifies SLC25A45 as necessary for transport of mitochondrial PR:PROJECT_TITLE methylated amino acids and carnitine synthesis PR:PROJECT_SUMMARY Solute carriers (SLCs) regulate cellular and organismal metabolism by PR:PROJECT_SUMMARY transporting small molecules and ions across membranes, yet the physiological PR:PROJECT_SUMMARY substrates of ~20% remain elusive. To address this, we developed a supervised PR:PROJECT_SUMMARY machine learning platform to predict and prioritize gene-metabolite PR:PROJECT_SUMMARY associations. We uncover SLC25A45 as a mitochondrial transporter linked to serum PR:PROJECT_SUMMARY levels of methylated basic amino acids, products of protein catabolism. PR:PROJECT_SUMMARY Mechanistically, SLC25A45 is necessary for the mitochondrial import of PR:PROJECT_SUMMARY methylated basic amino acids, including ADMA and TML, the latter serving as a PR:PROJECT_SUMMARY precursor for carnitine synthesis. In line with this observation, SLC25A45 loss PR:PROJECT_SUMMARY impairs hepatic carnitine synthesis and upregulation of carnitine-containing PR:PROJECT_SUMMARY metabolites under fasted conditions. By facilitating mitochondrial TML import, PR:PROJECT_SUMMARY SLC25A45 connects protein catabolism to carnitine production, sustaining PR:PROJECT_SUMMARY β-oxidation during fasting. Altogether, our study identifies putative PR:PROJECT_SUMMARY substrates for three SLCs and provides a resource for transporter PR:PROJECT_SUMMARY deorphanization. PR:INSTITUTE Rockefeller University PR:LAST_NAME Artem PR:FIRST_NAME Khan PR:ADDRESS 1230 York Ave, New York, NY, 10065 PR:EMAIL akhan01@rockefeller.edu PR:PHONE (212) 327-7874 #STUDY ST:STUDY_TITLE Serum profiling of SLC25A45 mice under different diet/fasting conditions ST:STUDY_SUMMARY SLC25A45 KO vs control mice have been exposed to either (a) high protein diet ST:STUDY_SUMMARY (b) regular diet, (c) 24 fasting, (d) ad libitum, (e) 24 fasting with carnitine ST:STUDY_SUMMARY supplementation in drinking water. Serum was collected. ST:INSTITUTE Rockefeller University ST:LAST_NAME Khan ST:FIRST_NAME Artem ST:ADDRESS 1230 York Ave, New York, NY, USA, 10065 ST:EMAIL akhan01@rockefeller.edu ST:PHONE (212) 327-7874 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - 83 KO Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_01.raw SUBJECT_SAMPLE_FACTORS - 71 KO Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_02.raw SUBJECT_SAMPLE_FACTORS - 69 KO Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_03.raw SUBJECT_SAMPLE_FACTORS - 42 KO Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_04.raw SUBJECT_SAMPLE_FACTORS - 33 KO Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_05.raw SUBJECT_SAMPLE_FACTORS - 57 WT Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_06.raw SUBJECT_SAMPLE_FACTORS - 55 WT Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_07.raw SUBJECT_SAMPLE_FACTORS - 45 WT Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_08.raw SUBJECT_SAMPLE_FACTORS - 41 WT Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_09.raw SUBJECT_SAMPLE_FACTORS - 31 WT Sample source:Serum | Diet:Ad lib RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_10.raw SUBJECT_SAMPLE_FACTORS - 73 KO Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_11.raw SUBJECT_SAMPLE_FACTORS - 91 KO Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_12.raw SUBJECT_SAMPLE_FACTORS - 90 KO Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_13.raw SUBJECT_SAMPLE_FACTORS - 107 KO Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_14.raw SUBJECT_SAMPLE_FACTORS - 64 KO Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_15.raw SUBJECT_SAMPLE_FACTORS - 77 WT Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_16.raw SUBJECT_SAMPLE_FACTORS - 76 WT Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_17.raw SUBJECT_SAMPLE_FACTORS - 60 HET Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_18.raw SUBJECT_SAMPLE_FACTORS - 59 WT Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_19.raw SUBJECT_SAMPLE_FACTORS - 32 WT Sample source:Serum | Diet:Fasting RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_20.raw SUBJECT_SAMPLE_FACTORS - 40 KO Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_21.raw SUBJECT_SAMPLE_FACTORS - 35 KO Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_22.raw SUBJECT_SAMPLE_FACTORS - 34 KO Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_23.raw SUBJECT_SAMPLE_FACTORS - 8 KO Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_24.raw SUBJECT_SAMPLE_FACTORS - 2 KO Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_25.raw SUBJECT_SAMPLE_FACTORS - 106 KO Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_26.raw SUBJECT_SAMPLE_FACTORS - 86 WT Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_27.raw SUBJECT_SAMPLE_FACTORS - 30 WT Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_28.raw SUBJECT_SAMPLE_FACTORS - 28 WT Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_29.raw SUBJECT_SAMPLE_FACTORS - 27 WT Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_30.raw SUBJECT_SAMPLE_FACTORS - 23 WT Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_31.raw SUBJECT_SAMPLE_FACTORS - 10 WT Sample source:Serum | Diet:Carnitine fast RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_32.raw SUBJECT_SAMPLE_FACTORS - 79 KO Sample source:Serum | Diet:Control diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_33.raw SUBJECT_SAMPLE_FACTORS - 53 KO Sample source:Serum | Diet:Control diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_34.raw SUBJECT_SAMPLE_FACTORS - 51 KO Sample source:Serum | Diet:Control diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_35.raw SUBJECT_SAMPLE_FACTORS - 118 KO Sample source:Serum | Diet:Control diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_36.raw SUBJECT_SAMPLE_FACTORS - 108 KO Sample source:Serum | Diet:Control diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_37.raw SUBJECT_SAMPLE_FACTORS - 133 HET Sample source:Serum | Diet:Control diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_38.raw SUBJECT_SAMPLE_FACTORS - 67 WT Sample source:Serum | Diet:Control Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_39.raw SUBJECT_SAMPLE_FACTORS - 61 HET Sample source:Serum | Diet:Control Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_40.raw SUBJECT_SAMPLE_FACTORS - 56 WT Sample source:Serum | Diet:Control Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_41.raw SUBJECT_SAMPLE_FACTORS - 48 KO Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_42.raw SUBJECT_SAMPLE_FACTORS - 43 KO Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_43.raw SUBJECT_SAMPLE_FACTORS - 29 KO Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_44.raw SUBJECT_SAMPLE_FACTORS - 9 KO Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_45.raw SUBJECT_SAMPLE_FACTORS - 6 KO Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_46.raw SUBJECT_SAMPLE_FACTORS - 105 KO Sample source:Serum | Diet:High Prot diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_47.raw SUBJECT_SAMPLE_FACTORS - 104 KO Sample source:Serum | Diet:High Prot diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_48.raw SUBJECT_SAMPLE_FACTORS - 132 HET Sample source:Serum | Diet:High Prot diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_49.raw SUBJECT_SAMPLE_FACTORS - 131 HET Sample source:Serum | Diet:High Prot diet RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_50.raw SUBJECT_SAMPLE_FACTORS - 44 WT Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_51.raw SUBJECT_SAMPLE_FACTORS - 24 WT Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_52.raw SUBJECT_SAMPLE_FACTORS - 22 WT Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_53.raw SUBJECT_SAMPLE_FACTORS - 17 WT Sample source:Serum | Diet:High Prot RAW_FILE_NAME(Raw file name)=MS259595IQM_artem_birsoy_54.raw SUBJECT_SAMPLE_FACTORS - - Sample source:- | Diet:- RAW_FILE_NAME(Raw file name)=- #COLLECTION CO:COLLECTION_SUMMARY Mice have been exposed to the indicated dietary conditions and the serum has CO:COLLECTION_SUMMARY been collected by bleeding mice cheek. Freshly collected blood was stored on ice CO:COLLECTION_SUMMARY (4C); then centrifuged for ~500 g for 5 min (4 C), and 5 uL of the supernatant CO:COLLECTION_SUMMARY was mixed with cold (4C) 45 uL of 75%AcN:25%MeOH extraction buffer, following CO:COLLECTION_SUMMARY mixing and centifugation at maximum speed at 4 C (>14,000 g). Supernatant was CO:COLLECTION_SUMMARY transferred. CO:SAMPLE_TYPE Blood (serum) CO:COLLECTION_TUBE_TEMP 4 #TREATMENT TR:TREATMENT_SUMMARY Mice have been placed on a vegetarian chow diet for ~2 weeks/high protein TR:TREATMENT_SUMMARY diet/control protein diet. The mice on vegetarian chow diet were subjected TR:TREATMENT_SUMMARY either 24 hour fasting with/without carnitine in the drinking water or ad TR:TREATMENT_SUMMARY libitum conditions. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Freshly collected blood was spin down for ~500 g for 5 min, and 5 uL of the SP:SAMPLEPREP_SUMMARY supernatant was mixed with 45 uL of 75%AcN:25%MeOH extraction buffer, following SP:SAMPLEPREP_SUMMARY mixing and centifugation at maximum speed at 4 C (>14,000 g). Supernatant was SP:SAMPLEPREP_SUMMARY transferred. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME EMD Millipore CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A 20 mM ammonium carbonate and 0.1% ammonium hydroxide in water at pH 9.3 CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT The gradient (vol/vol) used was as follows: 0–22 min, linear gradient from 90% CH:FLOW_GRADIENT to 40% B; 22–24 min, held at 40% B; 24–24.1 min, returned to 90% B; CH:FLOW_GRADIENT 24.1–30 min, equilibrated at 90% B at a flow rate of 150 μl/min. CH:FLOW_RATE flow rate of 150 μl/min. CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap IQ-X Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE EI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS The IQ-X Tribrid was operated in full-scan polarity-switching mode with a spray MS:MS_COMMENTS voltage of +3 kV and -2.5 kV, sheath gas at 40, auxiliary gas at 15, sweep gas MS:MS_COMMENTS at 1, an ion-transfer tube temperature of 275 °C, and a probe heater MS:MS_COMMENTS temperature of 350 °C. Samples were analyzed with a resolution of 120,000, an MS:MS_COMMENTS AGC target of 1.2 × 10⁵, a maximum injection time of 246 ms, and two mass MS:MS_COMMENTS ranges of 55–400 Th and 375–875 Th. Both mass spectrometers were externally MS:MS_COMMENTS calibrated every three days or weekly using Pierce Calibration Solution (Thermo MS:MS_COMMENTS Fisher Scientific). Sample injections were randomized. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS m/z MS_METABOLITE_DATA_START Samples 83 KO 71 KO 69 KO 42 KO 33 KO 57 WT 55 WT 45 WT 41 WT 31 WT 73 KO 91 KO 90 KO 107 KO 64 KO 77 WT 76 WT 60 HET 59 WT 32 WT 40 KO 35 KO 34 KO 8 KO 2 KO 106 KO 86 WT 30 WT 28 WT 27 WT 23 WT 10 WT 79 KO 53 KO 51 KO 118 KO 108 KO 133 HET 67 WT 61 HET 56 WT 48 KO 43 KO 29 KO 9 KO 6 KO 105 KO 104 KO 132 HET 131 HET 44 WT 24 WT 22 WT 17 WT Factors Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Ad lib Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Fasting Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Carnitine fast Sample source:Serum | Diet:Control diet Sample source:Serum | Diet:Control diet Sample source:Serum | Diet:Control diet Sample source:Serum | Diet:Control diet Sample source:Serum | Diet:Control diet Sample source:Serum | Diet:Control diet Sample source:Serum | Diet:Control Prot Sample source:Serum | Diet:Control Prot Sample source:Serum | Diet:Control Prot Sample source:Serum | Diet:High Prot Sample source:Serum | Diet:High Prot Sample source:Serum | Diet:High Prot Sample source:Serum | Diet:High Prot Sample source:Serum | Diet:High Prot Sample source:Serum | Diet:High Prot diet Sample source:Serum | Diet:High Prot diet Sample source:Serum | Diet:High Prot diet Sample source:Serum | Diet:High Prot diet Sample source:Serum | Diet:High Prot Sample source:Serum | Diet:High Prot Sample source:Serum | Diet:High Prot Sample source:Serum | Diet:High Prot acetylcarnitine 2314891008 4729019392 3755182592 2946562304 3676389888 2640909312 3257058816 4057097472 2937524992 2768199168 3716545536 5462223872 4983742976 4074579712 4343187456 6898506752 7447498752 11025022976 6912290304 6408449536 20920141824 13896059904 22325725184 18202767360 16246844416 16852721664 18260684800 17140244480 13614279680 20552235008 15493741568 16612115456 3250431232 3213316864 2776801024 2045518464 2595776768 4289465088 4622168576 3198067712 4122890496 2813585152 2326928128 2238719232 3564634368 2563269120 736090688 777398336 2093166976 1400965120 1896661504 3727152896 2604083200 2312334336 glutamine 8005413376 8393246720 8917365760 6696176640 8482227712 8770271232 8396120576 8127606272 8891283456 8477857792 8524846080 8333504000 9319106560 10130003968 7982379008 7388941312 8104996352 10153505792 8591597568 6674493440 8792946688 8326111744 9326768128 9084797952 8795553792 9412042752 8193200128 10631624704 7868249088 8479831552 8520076288 10898274304 8326258688 6174820864 7557655040 7706923008 9727910912 7397134848 8963764224 6940711424 6994580992 6074977280 10436423680 6669126656 8938215424 8460790784 12555754496 8437240320 8105472512 7641904128 7655988736 8591100928 6369649664 7621722112 propionylcarnitine 190766160 333276768 133281448 156566320 162446240 207079680 258462288 268980768 310900768 183902656 65715452 67681368 67500512 86177248 65664500 102705616 133775776 187590880 113536808 81459400 681010368 275394720 540959872 434687904 282766560 257467248 352749536 388693568 219282800 781983168 236312640 345840224 196414336 104206256 159239472 99368872 172033744 146623536 303845856 201514048 195680192 213828032 228000640 227671440 259088592 206262736 180574096 92145064 123224520 72882552 120845656 245006672 216190160 134357648 butyryl-carnitine 153157104 273736800 169935008 151570528 218055008 342520192 217221920 286971104 272994912 207877920 76274440 84504096 72401760 92553824 79244688 119336848 123246608 261993872 114434632 107017680 481814912 291240832 416510688 402851616 293265248 273648576 325342560 328629184 198396224 417961600 214467952 341489920 420180736 195124032 298318208 143473040 504023744 107312680 491925088 247390224 409987072 219518000 111227680 159078912 222218960 197526448 67138040 86366600 183351648 74562320 109985008 218456912 154870832 123999112 carnitine 4598181888 4902137344 3518643712 3126050048 3671458816 5707320320 4762770944 6291986944 6968265728 5195568128 1083338240 1601060352 1734397056 1369730176 1518938496 1662026240 1654081792 3310872320 2474673664 1575797120 8451631616 7067216384 8657445888 8507819008 4954987520 7675107328 11699228672 8040077824 9002438656 10500177920 6879410176 8401152000 4788222464 2864178176 3506537216 3057979648 4757562880 3658164224 5803680256 5627452928 4315716096 3007848192 3975714048 2754739968 3601574400 3390315008 1875366272 1873933056 4094968320 1923524480 2683051008 4042221312 3282308352 3883778304 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z retention index KEGG ID acetylcarnitine 204.123034 8.97 C02571 glutamine 147.076418 13.62 C00064 propionylcarnitine 218.138684 7.89 C03017 butyryl-carnitine 232.154334 7.09 C02862 carnitine 162.112469 11.25 C00318 METABOLITES_END #END