#METABOLOMICS WORKBENCH XiaoyangSu_20250619_134448 DATATRACK_ID:6065 STUDY_ID:ST004015 ANALYSIS_ID:AN006620 PROJECT_ID:PR002513
VERSION             	1
CREATED_ON             	June 19, 2025, 4:51 pm
#PROJECT
PR:PROJECT_TITLE                 	SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo
PR:PROJECT_TITLE                 	synthesis
PR:PROJECT_TYPE                  	Cellular polar metabolomics
PR:PROJECT_SUMMARY               	Solute carriers (SLC) are membrane proteins that facilitate the transportation
PR:PROJECT_SUMMARY               	of ions and metabolites across either the plasma membrane or the membrane of
PR:PROJECT_SUMMARY               	intracellular organelles. With more than 450 human genes annotated as SLCs, many
PR:PROJECT_SUMMARY               	of them are still orphan transporters without known biochemical functions. We
PR:PROJECT_SUMMARY               	developed a metabolomic-transcriptomic association analysis, and we found that
PR:PROJECT_SUMMARY               	the expression of SLC45A4 has a strong positive correlation with the cellular
PR:PROJECT_SUMMARY               	level of γ-aminobutyric acid (GABA). Using mass spectrometry and the stable
PR:PROJECT_SUMMARY               	isotope tracing approach, we demonstrated that SLC45A4 promotes GABA de novo
PR:PROJECT_SUMMARY               	synthesis through the Arginine/Ornithine/Putrescine (AOP) pathway. SLC45A4
PR:PROJECT_SUMMARY               	functions as a putrescine transporter localized to the peroxisome membrane to
PR:PROJECT_SUMMARY               	facilitate GABA production. Taken together, our results revealed a new
PR:PROJECT_SUMMARY               	biochemical mechanism where SLC45A4 controls GABA production.
PR:INSTITUTE                     	Rutgers University
PR:DEPARTMENT                    	Department of Medicine
PR:LAST_NAME                     	Su
PR:FIRST_NAME                    	Xiaoyang
PR:ADDRESS                       	7118 Clinical Academic Building
PR:EMAIL                         	xs137@rwjms.rutgers.edu
PR:PHONE                         	7322355447
PR:FUNDING_SOURCE                	R01 GM149664
#STUDY
ST:STUDY_TITLE                   	SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo
ST:STUDY_TITLE                   	synthesis
ST:STUDY_SUMMARY                 	We found that the expression of SLC45A4 has a strong positive correlation with
ST:STUDY_SUMMARY                 	the cellular level of γ-aminobutyric acid (GABA). Using mass spectrometry and
ST:STUDY_SUMMARY                 	the stable isotope tracing approach, we demonstrated that SLC45A4 promotes GABA
ST:STUDY_SUMMARY                 	de novo synthesis through the Arginine/Ornithine/Putrescine (AOP) pathway.
ST:STUDY_SUMMARY                 	SLC45A4 functions as a putrescine transporter localized to the peroxisome
ST:STUDY_SUMMARY                 	membrane to facilitate GABA production. Taken together, our results revealed a
ST:STUDY_SUMMARY                 	new biochemical mechanism where SLC45A4 controls GABA production.
ST:INSTITUTE                     	Rutgers University
ST:DEPARTMENT                    	Department of Medicine
ST:LAST_NAME                     	Su
ST:FIRST_NAME                    	Xiaoyang
ST:ADDRESS                       	7118 Clinical Academic Building
ST:EMAIL                         	xs137@rwjms.rutgers.edu
ST:PHONE                         	17322355447
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:CELL_STRAIN_DETAILS           	A549, H1299
#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           	-	lysate_A549_KD45A4_1	Sample source:Lysate | Cell line:A549 | Knockdown:SLC45A4	RAW_FILE_NAME(Raw file name)=lysate_A549_KD45A4_1.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_A549_KD45A4_2	Sample source:Lysate | Cell line:A549 | Knockdown:SLC45A4	RAW_FILE_NAME(Raw file name)=lysate_A549_KD45A4_2.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_A549_KD45A4_3	Sample source:Lysate | Cell line:A549 | Knockdown:SLC45A4	RAW_FILE_NAME(Raw file name)=lysate_A549_KD45A4_3.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_A549_siCtr_1	Sample source:Lysate | Cell line:A549 | Knockdown:Ctrl	RAW_FILE_NAME(Raw file name)=lysate_A549_siCtr_1.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_A549_siCtr_2	Sample source:Lysate | Cell line:A549 | Knockdown:Ctrl	RAW_FILE_NAME(Raw file name)=lysate_A549_siCtr_2.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_A549_siCtr_3	Sample source:Lysate | Cell line:A549 | Knockdown:Ctrl	RAW_FILE_NAME(Raw file name)=lysate_A549_siCtr_3.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_H1299_KD45A4_1	Sample source:Lysate | Cell line:H1299 | Knockdown:SLC45A4	RAW_FILE_NAME(Raw file name)=lysate_H1299_KD45A4_1.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_H1299_KD45A4_2	Sample source:Lysate | Cell line:H1299 | Knockdown:SLC45A4	RAW_FILE_NAME(Raw file name)=lysate_H1299_KD45A4_2.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_H1299_KD45A4_3	Sample source:Lysate | Cell line:H1299 | Knockdown:SLC45A4	RAW_FILE_NAME(Raw file name)=lysate_H1299_KD45A4_3.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_H1299_siCtr_1	Sample source:Lysate | Cell line:H1299 | Knockdown:Ctrl	RAW_FILE_NAME(Raw file name)=lysate_H1299_siCtr_1.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_H1299_siCtr_2	Sample source:Lysate | Cell line:H1299 | Knockdown:Ctrl	RAW_FILE_NAME(Raw file name)=lysate_H1299_siCtr_2.mzXML
SUBJECT_SAMPLE_FACTORS           	-	lysate_H1299_siCtr_3	Sample source:Lysate | Cell line:H1299 | Knockdown:Ctrl	RAW_FILE_NAME(Raw file name)=lysate_H1299_siCtr_3.mzXML
#COLLECTION
CO:COLLECTION_SUMMARY            	0.5×10^6 cells in the culture were washed twice with PBS and extracted with 0.5
CO:COLLECTION_SUMMARY            	ml ice-cold 40:40:20 (methanol:acetonitrile:water) solution with 0.5% formic
CO:COLLECTION_SUMMARY            	acid. The cells were scraped off the plate followed by incubation on ice for 10
CO:COLLECTION_SUMMARY            	min, and neutralized by the addition of 25 μl 15% (m/v) NH4HCO3 solution. The
CO:COLLECTION_SUMMARY            	extracts were transferred to a 1.5 ml tube. For conditioned media metabolite
CO:COLLECTION_SUMMARY            	extraction, 5 µl of culture media was mixed with 500 µl of ice-cold 40:40:20
CO:COLLECTION_SUMMARY            	(methanol:acetonitrile:water) solution with 0.5% formic acid and neutralized by
CO:COLLECTION_SUMMARY            	the addition of 25 μl 15% (m/v) NH4HCO3 solution. The whole cell or media
CO:COLLECTION_SUMMARY            	extracts were then centrifuged at 4°C and 16,000 × g for 10 min and
CO:COLLECTION_SUMMARY            	transferred to a clean tube and stored at -80°C until analysis.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	A549 or H1299 cells were seeded in 6-well plates at the density of 2×105 / ml.
TR:TREATMENT_SUMMARY             	When the cells reached about 70% confluence, cells were transfected with siRNA
TR:TREATMENT_SUMMARY             	Transfection Reagent (Santa Cruz Biotechnology, sc-29528) according to
TR:TREATMENT_SUMMARY             	manufacturer’s instructions. siRNAs targeting human SLC45A4 (SCBT, sc-77846)
TR:TREATMENT_SUMMARY             	and non-targeting Scramble controls (SCBT, sc-37007) were obtained from SCBT and
TR:TREATMENT_SUMMARY             	re-suspended at 10 μM in RNAse free water. 4 μl siRNA (20 nM) was first mixed
TR:TREATMENT_SUMMARY             	with 100 μl Opti-MEM (Gibco, 31985070), then mixed with 4 μl siRNA
TR:TREATMENT_SUMMARY             	Transfection Reagent contained in another 100 μl Opti-MEM. The mixture was
TR:TREATMENT_SUMMARY             	incubated at room temperature for 30 min and mixed with 800 μl Opti-MEM. Cells
TR:TREATMENT_SUMMARY             	were washed once with 2 ml Opti-MEM, then covered with 1 ml siRNA mixture and
TR:TREATMENT_SUMMARY             	incubated for 6 hours at 37 °C, 5% CO2. After the incubation, 1 ml of RPMI
TR:TREATMENT_SUMMARY             	1640 media with 20% FBS and 2× Penicillin-Streptomycin were added.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The whole cell extracts were then centrifuged at 4°C and 16,000 × g for 10 min
SP:SAMPLEPREP_SUMMARY            	and transferred to a clean tube and stored at -80°C until analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters XBridge BEH Amide (150 mm × 2.1 mm, 2.5um)
CH:SOLVENT_A                     	95%:5% H2O:acetonitrile with 20 mM acetic acid, 40 mM ammonium hydroxide, pH 9.4
CH:SOLVENT_B                     	0%:80% H2O:acetonitrile with 20 mM acetic acid, 40 mM ammonium hydroxide, pH 9.4
CH:FLOW_GRADIENT                 	0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B;
CH:FLOW_GRADIENT                 	10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45%
CH:FLOW_GRADIENT                 	B; 16.5 min, 100% B; and 22 min, 100% B
CH:FLOW_RATE                     	0.3ml/min
CH:COLUMN_TEMPERATURE            	25
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Metabolomics Shared Resource of Rutgers Cancer Institute
AN:DETECTOR_TYPE                 	Orbitrap
AN:DATA_FORMAT                   	mzXML
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Plus Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The mass spectrometry analysis was performed on Thermo Q Exactive PLUS
MS:MS_COMMENTS                   	instrument with a HESI source, which was set to a spray voltage of −2.7 kV in
MS:MS_COMMENTS                   	negative mode and 3.5 kV in positive mode. The sheath, auxiliary, and sweep gas
MS:MS_COMMENTS                   	flow rates were 40, 10, and 2 (arbitrary units), respectively. The capillary
MS:MS_COMMENTS                   	temperature was set to 300°C, and the aux gas heater was set to 360°C. The
MS:MS_COMMENTS                   	S-lens RF level was 45. The m/z scan range was set to 72 to 1000 m/z in either
MS:MS_COMMENTS                   	positive or negative ionization mode. The AGC target was set to 3e6, and the
MS:MS_COMMENTS                   	maximum IT was 200 ms. Metabolite data were obtained using the El-MAVEN software
MS:MS_COMMENTS                   	package (mass accuracy window: 5 ppm). All isotope natural abundance corrections
MS:MS_COMMENTS                   	were done using AccuCor (single-isotope labeling) or AccuCor2 (dual-isotope
MS:MS_COMMENTS                   	labeling).
MS:CAPILLARY_TEMPERATURE         	300C
MS:ION_SPRAY_VOLTAGE             	3.5kV
MS:MS_RESULTS_FILE               	ST004015_AN006620_Results.txt	UNITS:Ion counts	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END