#METABOLOMICS WORKBENCH yohei57jp_20241209_011357 DATATRACK_ID:5444 STUDY_ID:ST003626 ANALYSIS_ID:AN005957 PROJECT_ID:PR002239
VERSION             	1
CREATED_ON             	December 12, 2024, 5:31 pm
#PROJECT
PR:PROJECT_TITLE                 	Metabolomic profile of human regulatory T cells.
PR:PROJECT_SUMMARY               	Regulatory T cells (Tregs) are characterized by stable FOXP3 expression and
PR:PROJECT_SUMMARY               	controlling immune response by suppressive activity. Unique metabolic properties
PR:PROJECT_SUMMARY               	of Tregs are shown such as reduced glycolysis and increased oxidative
PR:PROJECT_SUMMARY               	phosphorylation. We have combined transcriptomics, metabolomics and lipidomics
PR:PROJECT_SUMMARY               	to dissect metabolic dynamics of Tregs upon activation. Combined metabolomic and
PR:PROJECT_SUMMARY               	lipidomic analysis showed that freshly isolated Tregs have unique metabolomic
PR:PROJECT_SUMMARY               	property, on the other hand, activated Tregs have unique lipidomic property.
PR:PROJECT_SUMMARY               	Interestingly, activated Tregs contained omega-3 polyunsaturated fatty acids
PR:PROJECT_SUMMARY               	(PUFA) enriched diglycerides and triglycerides.
PR:INSTITUTE                     	The Jikei University School of Medicine
PR:LAST_NAME                     	Sato
PR:FIRST_NAME                    	Yohei
PR:ADDRESS                       	3-25-8 Nishishinbashi
PR:EMAIL                         	yoheisatoo@gmail.com
PR:PHONE                         	+81-3-3433-1111
#STUDY
ST:STUDY_TITLE                   	Metabolomic profile of human regulatory T cells.
ST:STUDY_SUMMARY                 	Regulatory T cells (Tregs) are characterized by stable FOXP3 expression and
ST:STUDY_SUMMARY                 	controlling immune response by suppressive activity. Unique metabolic properties
ST:STUDY_SUMMARY                 	of Tregs are shown such as reduced glycolysis and increased oxidative
ST:STUDY_SUMMARY                 	phosphorylation. We have combined transcriptomics, metabolomics and lipidomics
ST:STUDY_SUMMARY                 	to dissect metabolic dynamics of Tregs upon activation. Combined metabolomic and
ST:STUDY_SUMMARY                 	lipidomic analysis showed that freshly isolated Tregs have unique metabolomic
ST:STUDY_SUMMARY                 	property, on the other hand, activated Tregs have unique lipidomic property.
ST:STUDY_SUMMARY                 	Interestingly, activated Tregs contained omega-3 polyunsaturated fatty acids
ST:STUDY_SUMMARY                 	(PUFA) enriched diglycerides and triglycerides.
ST:INSTITUTE                     	The Jikei University School of Medicine
ST:LAST_NAME                     	Sato
ST:FIRST_NAME                    	Yohei
ST:ADDRESS                       	3-25-8 Nishishinbashi
ST:EMAIL                         	yoheisatoo@gmail.com
ST:PHONE                         	+81-3-3433-1111
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#FACTORS
#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           	-	Teff1	Group:T-cells(Teff) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Teff2	Group:T-cells(Teff) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Teff3	Group:T-cells(Teff) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Treg1	Group:T-cells(Treg) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Treg2	Group:T-cells(Treg) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Treg3	Group:T-cells(Treg) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Activated Teff1	Group:Activated T-cells(Teff) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Activated Teff2	Group:Activated T-cells(Teff) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Activated Teff3	Group:Activated T-cells(Teff) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Activated Treg1	Group:Activated T-cells(Treg) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Activated Treg2	Group:Activated T-cells(Treg) | Sample source:-	
SUBJECT_SAMPLE_FACTORS           	-	Activated Treg3	Group:Activated T-cells(Treg) | Sample source:-	
#COLLECTION
CO:COLLECTION_SUMMARY            	Human regulatory T cells were sorted according to the CD4+CD25+CD127-
CO:COLLECTION_SUMMARY            	populations by flow cytometry. Human effector T cells were sorted according to
CO:COLLECTION_SUMMARY            	the CD4+CD25-CD127+ populations by flow cytometry. Cells were directly sorted in
CO:COLLECTION_SUMMARY            	RPMI medium supplemented with 10% FBS. Spin down the collected cells and cell
CO:COLLECTION_SUMMARY            	pellets were stored at -80C.
CO:SAMPLE_TYPE                   	T-cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Human regulatory T cells were sorted and activated overnight by CD3/CD28/CD2
TR:TREATMENT_SUMMARY             	stimulation in the presence of 100U/ml recombinant human IL-2.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	After culturing the regulatory T cells, the medium was removed and washed twice
SP:SAMPLEPREP_SUMMARY            	with mannitol (WAKO pure chemical). A methanol (WAKO pure chemical) solution was
SP:SAMPLEPREP_SUMMARY            	added and stirred. Milli-Q water containing 10 μM of an internal standard (HMT,
SP:SAMPLEPREP_SUMMARY            	Patent No. 6173667) was added and stirred, and centrifuged (2,300 × g, 4°C, 5
SP:SAMPLEPREP_SUMMARY            	min) (Patent No. 6173667). After centrifugation, the extract was transferred to
SP:SAMPLEPREP_SUMMARY            	an ultrafiltration tube (Ultrafree MC PLHCC, HMT, centrifugal filter unit 5
SP:SAMPLEPREP_SUMMARY            	kDa). This was centrifuged (9,100 × g, 4°C) and subjected to ultrafiltration.
SP:SAMPLEPREP_SUMMARY            	The filtrate was dried and dissolved in Milli-Q water again for measurement.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	CE
CH:INSTRUMENT_NAME               	Agilent 7100 CE
CH:COLUMN_NAME                   	Fused silica capillary i.d. 50 um x 80cm
CH:SOLVENT_A                     	Anion Buffer Solution(p/n:I3302-1023)
CH:SOLVENT_B                     	N/A
CH:FLOW_GRADIENT                 	N/A
CH:FLOW_RATE                     	N/A
CH:COLUMN_TEMPERATURE            	N/A
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6230 TOF
MS:INSTRUMENT_TYPE               	TOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	CE-TOFMS(Basic) negative(Anion) mode. The detected peaks were matched and
MS:MS_COMMENTS                   	searched against all substances registered in the HMT metabolite library based
MS:MS_COMMENTS                   	on the m/z and MT values. The search tolerance was set to ± 0.5 min for MT and
MS:MS_COMMENTS                   	± 10 ppm for m/z. Mass error (ppm) = (Actual value – theoretical value)/
MS:MS_COMMENTS                   	Actual value×106 For substances with molecular weights of 100 or less and some
MS:MS_COMMENTS                   	substances expected to be present in large amounts in the sample, the tolerance
MS:MS_COMMENTS                   	was set wider than the above conditions and the search was performed. In
MS:MS_COMMENTS                   	addition, if the candidates could not be narrowed down and the same candidate
MS:MS_COMMENTS                   	metabolite was assigned to multiple peaks, a branch number was added and the
MS:MS_COMMENTS                   	list was displayed. The target metabolites were analyzed. The calibration curve
MS:MS_COMMENTS                   	was made using peak areas corrected by the internal standard substance, and the
MS:MS_COMMENTS                   	concentration was calculated for each substance with a single calibration of 100
MS:MS_COMMENTS                   	μM (internal standard substance 200 μM).
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	arbitrary number
MS_METABOLITE_DATA_START
Samples	Teff1	Teff2	Teff3	Treg1	Treg2	Treg3	Activated Teff1	Activated Teff2	Activated Teff3	Activated Treg1	Activated Treg2	Activated Treg3
Factors	Group:T-cells(Teff) | Sample source:-	Group:T-cells(Teff) | Sample source:-	Group:T-cells(Teff) | Sample source:-	Group:T-cells(Treg) | Sample source:-	Group:T-cells(Treg) | Sample source:-	Group:T-cells(Treg) | Sample source:-	Group:Activated T-cells(Teff) | Sample source:-	Group:Activated T-cells(Teff) | Sample source:-	Group:Activated T-cells(Teff) | Sample source:-	Group:Activated T-cells(Treg) | Sample source:-	Group:Activated T-cells(Treg) | Sample source:-	Group:Activated T-cells(Treg) | Sample source:-
Lactic acid	2.7E+00	2.7E+00	2.6E+00	3.1E+00	3.2E+00	3.3E+00	1.6E+00	1.6E+00	1.7E+00	1.6E+00	1.6E+00	1.6E+00
3-Hydroxybutyric acid	1.8E-02	N.D.	N.D.	N.D.	N.D.	N.D.	7.5E-03	6.8E-03	8.8E-03	7.8E-03	8.3E-03	7.3E-03
Succinic acid	1.2E-01	1.2E-01	1.3E-01	1.5E-01	1.6E-01	1.5E-01	5.4E-02	5.1E-02	5.7E-02	5.3E-02	5.1E-02	5.1E-02
2-Hydroxyvaleric acid"	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	2.1E-03
Isethionic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	2.0E-03	2.0E-03	2.5E-03	2.0E-03	2.4E-03	2.9E-03
5-Oxoproline	2.5E+00	2.5E+00	2.5E+00	3.2E+00	3.3E+00	3.4E+00	7.6E-01	7.3E-01	7.8E-01	7.4E-01	7.4E-01	7.4E-01
Itaconic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	3.6E-03	N.D.	N.D.
2-Oxohexanoic acid"	2.4E-02	N.D.	N.D.	3.9E-02	N.D.	N.D.	9.6E-03	9.2E-03	9.9E-03	9.8E-03	9.3E-03	N.D.
Iminodiacetic acid	2.6E-01	3.0E-01	2.6E-01	3.3E-01	3.3E-01	3.5E-01	1.1E-01	1.1E-01	1.2E-01	1.2E-01	1.1E-01	1.2E-01
Malic acid	5.6E-02	4.8E-02	4.8E-02	5.3E-02	6.3E-02	6.8E-02	2.4E-02	2.7E-02	2.9E-02	2.5E-02	2.5E-02	2.7E-02
Threonic acid	5.0E-02	5.1E-02	5.0E-02	6.1E-02	6.1E-02	5.8E-02	1.8E-02	1.9E-02	1.9E-02	1.8E-02	1.8E-02	1.9E-02
Ethanolamine phosphate	8.4E-02	8.2E-02	7.3E-02	1.2E-01	1.2E-01	1.1E-01	2.9E-02	2.9E-02	3.0E-02	3.4E-02	3.6E-02	3.6E-02
2-Ethylhexanoic acid	7.1E-03	N.D.	7.7E-03	N.D.	N.D.	N.D.	3.3E-03	3.7E-03	3.1E-03	3.2E-03	N.D.	3.4E-03
N-Propionylalanine"	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1.1E-03	1.8E-03	1.4E-03	9.3E-04	1.1E-03	N.D.
3-Phenylpropionic acid	N.D.	7.3E-03	7.4E-03	N.D.	N.D.	N.D.	4.0E-03	3.1E-03	4.5E-03	3.9E-03	4.1E-03	3.7E-03
2-(Hydroxymethyl)benzoic acid"	4.1E-02	4.1E-02	4.4E-02	4.2E-02	4.5E-02	4.6E-02	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
Pelargonic acid	N.D.	8.1E-03	N.D.	N.D.	N.D.	N.D.	2.3E-03	2.4E-03	3.2E-03	2.7E-03	3.1E-03	2.4E-03
Terephthalic acid	7.2E-03	8.3E-03	8.7E-03	1.0E-02	1.1E-02	N.D.	2.6E-03	1.9E-03	2.5E-03	1.9E-03	N.D.	1.8E-03
Arabinonic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1.5E-03	N.D.	1.6E-03	1.9E-03	N.D.
N-Acetyltaurine	N.D.	6.2E-03	N.D.	N.D.	6.4E-03	N.D.	2.0E-03	2.0E-03	2.2E-03	1.7E-03	2.0E-03	2.0E-03
Uric acid	8.1E-03	7.5E-03	7.7E-03	1.0E-02	1.2E-02	1.2E-02	1.9E-03	1.7E-03	2.1E-03	2.3E-03	2.0E-03	2.2E-03
Dihydroxyacetone phosphate	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1.6E-03	1.7E-03	N.D.	2.2E-03	1.9E-03	2.3E-03
Glycerol 3-phosphate	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1.6E-03	1.5E-03	N.D.	1.5E-03	2.1E-03	1.6E-03
Decanoic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	2.1E-03	N.D.	N.D.	2.3E-03	2.4E-03	N.D.
cis-Aconitic acid	1.3E-02	N.D.	N.D.	N.D.	N.D.	N.D.	5.2E-03	5.7E-03	5.3E-03	5.6E-03	6.1E-03	5.4E-03
Allantoic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1.4E-03	1.8E-03	N.D.	1.4E-03	1.6E-03	1.6E-03
Hippuric acid	2.6E-02	2.5E-02	2.1E-02	2.7E-02	2.8E-02	2.8E-02	8.6E-03	1.0E-02	9.4E-03	7.8E-03	8.7E-03	1.0E-02
3-Phosphoglyceric acid	3.2E-02	2.6E-02	3.1E-02	4.4E-02	4.1E-02	4.4E-02	5.6E-03	5.5E-03	4.6E-03	5.8E-03	5.4E-03	5.8E-03
p-Cresol sulfate	6.9E-03	7.0E-03	9.0E-03	N.D.	1.0E-02	7.9E-03	2.5E-03	2.6E-03	3.0E-03	2.5E-03	2.7E-03	3.2E-03
Isocitric acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	6.1E-03	6.2E-03	5.5E-03	4.6E-03	N.D.
Citric acid	4.4E-01	4.9E-01	4.6E-01	5.3E-01	5.7E-01	5.8E-01	1.7E-01	1.7E-01	1.7E-01	1.7E-01	1.8E-01	1.6E-01
Quinic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1.6E-03	1.5E-03	1.5E-03
Phenaceturic acid"	3.7E-02	3.7E-02	4.1E-02	4.4E-02	3.7E-02	3.8E-02	1.7E-02	1.7E-02	1.7E-02	1.6E-02	1.7E-02	1.6E-02
Gluconic acid	N.D.	7.0E-03	N.D.	N.D.	N.D.	N.D.	2.4E-03	2.7E-03	2.5E-03	N.D.	N.D.	2.6E-03
Lauric acid	1.1E-02	9.0E-03	1.1E-02	9.9E-03	1.6E-02	1.6E-02	6.6E-03	9.2E-03	6.5E-03	6.9E-03	9.8E-03	6.7E-03
Pantothenic acid	1.4E-02	1.4E-02	1.3E-02	1.6E-02	1.3E-02	1.4E-02	6.1E-03	5.1E-03	4.9E-03	5.0E-03	6.3E-03	4.9E-03
Myristic acid	7.7E-03	7.5E-03	6.8E-03	N.D.	N.D.	1.0E-02	1.9E-03	3.4E-03	2.6E-03	2.5E-03	2.5E-03	2.5E-03
Biotin	N.D.	N.D.	N.D.	5.7E-03	N.D.	N.D.	1.6E-03	1.6E-03	N.D.	N.D.	N.D.	1.4E-03
Glucose 6-phosphate	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	2.2E-03	1.6E-03	2.1E-03	2.4E-03	1.9E-03	2.0E-03
Sulfotyrosine	4.9E-03	6.0E-03	6.0E-03	N.D.	6.1E-03	5.8E-03	2.0E-03	1.5E-03	1.7E-03	1.1E-03	2.0E-03	2.3E-03
6-Phosphogluconic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1.6E-03	N.D.	2.3E-03	1.3E-03	1.5E-03	N.D.
N-Acetylmuramic acid	1.2E-02	1.5E-02	1.3E-02	8.6E-03	1.7E-02	1.8E-02	2.7E-03	1.6E-03	2.4E-03	2.2E-03	2.3E-03	2.4E-03
Fructose 1,6-diphosphate	2.8E-02	3.1E-02	3.5E-02	4.8E-02	4.7E-02	5.1E-02	1.3E-02	1.3E-02	1.3E-02	1.4E-02	1.4E-02	1.4E-02
AMP	5.8E-03	6.7E-03	6.0E-03	N.D.	N.D.	N.D.	5.4E-03	5.9E-03	5.8E-03	6.7E-03	6.3E-03	6.5E-03
IMP	5.1E-03	7.4E-03	N.D.	N.D.	6.5E-03	N.D.	2.9E-03	2.3E-03	2.8E-03	3.5E-03	3.6E-03	3.3E-03
UDP	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	2.3E-03	2.2E-03	1.9E-03	2.3E-03	2.1E-03	2.2E-03
ADP	1.5E-02	1.7E-02	2.0E-02	2.2E-02	2.4E-02	2.3E-02	1.1E-02	1.0E-02	1.1E-02	1.3E-02	1.3E-02	1.3E-02
GDP	N.D.	N.D.	N.D.	6.7E-03	7.8E-03	6.7E-03	1.7E-03	1.8E-03	1.7E-03	1.7E-03	2.2E-03	2.0E-03
UTP	9.0E-03	N.D.	1.0E-02	1.2E-02	1.4E-02	1.6E-02	3.6E-03	2.3E-03	3.8E-03	3.4E-03	3.4E-03	2.9E-03
ATP	4.5E-02	4.3E-02	4.2E-02	6.1E-02	5.3E-02	6.0E-02	1.9E-02	1.8E-02	1.7E-02	1.9E-02	1.7E-02	1.8E-02
GTP	8.5E-03	N.D.	9.7E-03	1.3E-02	1.1E-02	1.4E-02	2.2E-03	2.2E-03	1.9E-03	1.9E-03	2.6E-03	2.0E-03
ADP-ribose	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1.0E-03	N.D.	1.5E-03	1.6E-03	1.8E-03	1.3E-03
UDP-glucose"	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	2.2E-03	2.8E-03	2.7E-03	2.2E-03	2.1E-03	2.6E-03
UDP-N-acetylgalactosamine"	N.D.	N.D.	N.D.	8.3E-03	7.3E-03	8.7E-03	2.2E-03	1.5E-03	2.0E-03	2.0E-03	2.0E-03	1.9E-03
NAD+	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	2.0E-03	1.7E-03	1.7E-03	1.6E-03	1.3E-03	1.6E-03			
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
Lactic acid
3-Hydroxybutyric acid
Succinic acid
2-Hydroxyvaleric acid"
Isethionic acid
5-Oxoproline
Itaconic acid
2-Oxohexanoic acid"
Iminodiacetic acid
Malic acid
Threonic acid
Ethanolamine phosphate
2-Ethylhexanoic acid
N-Propionylalanine"
3-Phenylpropionic acid
2-(Hydroxymethyl)benzoic acid"
Pelargonic acid
Terephthalic acid
Arabinonic acid
N-Acetyltaurine
Uric acid
Dihydroxyacetone phosphate
Glycerol 3-phosphate
Decanoic acid
cis-Aconitic acid
Allantoic acid
Hippuric acid
3-Phosphoglyceric acid
p-Cresol sulfate
Isocitric acid
Citric acid
Quinic acid
Phenaceturic acid"
Gluconic acid
Lauric acid
Pantothenic acid
Myristic acid
Biotin
Glucose 6-phosphate
Sulfotyrosine
6-Phosphogluconic acid
N-Acetylmuramic acid
Fructose 1,6-diphosphate
AMP
IMP
UDP
ADP
GDP
UTP
ATP
GTP
ADP-ribose
UDP-glucose"
UDP-N-acetylgalactosamine"
NAD+
METABOLITES_END
#END