#METABOLOMICS WORKBENCH akhan01_20250719_111746 DATATRACK_ID:6198 STUDY_ID:ST004054 ANALYSIS_ID:AN006701 PROJECT_ID:PR002544
VERSION             	1
CREATED_ON             	July 21, 2025, 1:55 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                   	Machine learning identifies SLC25A45 as necessary for transport of mitochondrial
ST:STUDY_TITLE                   	methylated amino acids and carnitine synthesis
ST:STUDY_SUMMARY                 	Metabolomics profiling of mitochondria purified from SLC25A45 KO vs control HeLa
ST:STUDY_SUMMARY                 	cells to determine the levels of methylated basic amino acids. Mitochondria
ST:STUDY_SUMMARY                 	purified from the KO cells contained less methylated basic amino acids (ADMA and
ST:STUDY_SUMMARY                 	TML) compared to the control ones, consistent with the role of SLC25A45 as
ST:STUDY_SUMMARY                 	necessary for mito transport of methylated basic amino acids.
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                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	Sa_1	Sample source:HeLa | Genotype:25A45_KO	RAW_FILE_NAME(Raw file name)=MS248897IQM_artem_birsoy_01.raw
SUBJECT_SAMPLE_FACTORS           	-	Sa_2	Sample source:HeLa | Genotype:25A45_KO	RAW_FILE_NAME(Raw file name)=MS248897IQM_artem_birsoy_02.raw
SUBJECT_SAMPLE_FACTORS           	-	Sa_3	Sample source:HeLa | Genotype:25A45_KO	RAW_FILE_NAME(Raw file name)=MS248897IQM_artem_birsoy_03.raw
SUBJECT_SAMPLE_FACTORS           	-	Sa_4	Sample source:HeLa | Genotype:Control	RAW_FILE_NAME(Raw file name)=MS248897IQM_artem_birsoy_04.raw
SUBJECT_SAMPLE_FACTORS           	-	Sa_5	Sample source:HeLa | Genotype:Control	RAW_FILE_NAME(Raw file name)=MS248897IQM_artem_birsoy_05.raw
SUBJECT_SAMPLE_FACTORS           	-	Sa_6	Sample source:HeLa | Genotype:Control	RAW_FILE_NAME(Raw file name)=MS248897IQM_artem_birsoy_06.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Mitochondria were immunopurified from ~10 mln HeLa cells expressing MITO-TAG by
CO:COLLECTION_SUMMARY            	using the magnetic beads conjugated to HA-Antibody. Following three washes in
CO:COLLECTION_SUMMARY            	KPBS, 50 μL 80% MeOH were added onto the beads to extract metabolites from the
CO:COLLECTION_SUMMARY            	immunopurified mitochondria. Samples were stored at -80°C before loading onto
CO:COLLECTION_SUMMARY            	LC-MS.
CO:SAMPLE_TYPE                   	HeLa cells
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatment has been done, baseline conditions
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	80% MeOH extract of immunopurified mitochondria were centrifuged at 4C, max
SP:SAMPLEPREP_SUMMARY            	speed (>14000 g), supernatant transferred into a vial for the LC-MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	EMD Millipore
CH:COLUMN_NAME                   	SeQuant ZIC- pHILIC (150 x 2.1 mm, 5 μm)
CH:SOLVENT_A                     	100% Water; 20 mM Ammonium carbonate; 0.1% Ammonium hydroxide (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°C
#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	Sa_1	Sa_2	Sa_3	Sa_4	Sa_5	Sa_6
Factors	Sample source:HeLa | Genotype:25A45_KO	Sample source:HeLa | Genotype:25A45_KO	Sample source:HeLa | Genotype:25A45_KO	Sample source:HeLa | Genotype:Control	Sample source:HeLa | Genotype:Control	Sample source:HeLa | Genotype:Control
ADMA	63368	74904	84343	362678	375773	234075
NAD	74854080	59890404	67363464	70338192	67948240	51369448
TML	81750	109177	88322	953201	889520	574692
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	retention index	quantified m/z	KEGG ID
ADMA	15.92	203.150252	C03626
NAD	16.81	664.116399	C00003
TML	14.36	189.15975	C03793
METABOLITES_END
#END