#METABOLOMICS WORKBENCH thejoeyli_20250612_100014 DATATRACK_ID:6033 STUDY_ID:ST004027 ANALYSIS_ID:AN006653 PROJECT_ID:PR002519
VERSION             	1
CREATED_ON             	June 12, 2025, 4:23 pm
#PROJECT
PR:PROJECT_TITLE                 	Primary human and mouse NK cell metabolomics
PR:PROJECT_SUMMARY               	Primary PBMC-derived human NK cells and splenic mouse NK cells were isolated and
PR:PROJECT_SUMMARY               	either immediately extracted for metabolites or cultured for 3 days in IL-2/15
PR:PROJECT_SUMMARY               	before metabolite extraction. Cells were rinsed with ice-cold 150 mM NH4AcO, pH
PR:PROJECT_SUMMARY               	7.3, resuspended in 80% ice-cold MeOH extraction buffer, and centrifuged for 10
PR:PROJECT_SUMMARY               	min at 17,000 g. Supernatants were collected and evaporated using a Nitrogen
PR:PROJECT_SUMMARY               	evaporator (Organomation). Evaporated extracts were stored at -80°C. Dried
PR:PROJECT_SUMMARY               	metabolites were re-suspended in 100 µL (1:1) dH20, ACN solution. 10% of these
PR:PROJECT_SUMMARY               	suspensions were injected per analysis. Samples were run on a Vanquish (Thermo
PR:PROJECT_SUMMARY               	Scientific) UHPLC system with mobile phase A (20 mM ammonium carbonate, pH 9.7)
PR:PROJECT_SUMMARY               	and mobile phase B (100% ACN) at a flow rate of 150 µL/min on a SeQuant
PR:PROJECT_SUMMARY               	ZIC-pHILIC Polymeric column (2.1 × 150 mm x 5 μm, EMD Millipore) at 35°C.
PR:PROJECT_SUMMARY               	Separation is achieved with a linear gradient from 20% A to 80% A in 20 min
PR:PROJECT_SUMMARY               	followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A
PR:PROJECT_SUMMARY               	is then held from 20.5 min to 28 min. The UHPLC is coupled to a Q-Exactive
PR:PROJECT_SUMMARY               	(Thermo Scientific) mass analyzer running in polarity switching mode at 3.2 kV
PR:PROJECT_SUMMARY               	with an MS1 resolution of 70,000.
PR:INSTITUTE                     	University of California, Los Angeles
PR:DEPARTMENT                    	MIMG
PR:LAST_NAME                     	Li
PR:FIRST_NAME                    	Joey
PR:ADDRESS                       	615 Charles E Young Dr S, Los Angeles CA 90095
PR:EMAIL                         	joeyhli@g.ucla.edu
PR:PHONE                         	4086213407
#STUDY
ST:STUDY_TITLE                   	Primary human and mouse NK cell metabolomics
ST:STUDY_SUMMARY                 	Primary PBMC-derived human NK cells and splenic mouse NK cells were isolated and
ST:STUDY_SUMMARY                 	either immediately extracted for metabolites or cultured for 3 days in IL-2/15
ST:STUDY_SUMMARY                 	before metabolite extraction. Cells were rinsed with ice-cold 150 mM NH4AcO, pH
ST:STUDY_SUMMARY                 	7.3, resuspended in 80% ice-cold MeOH extraction buffer, and centrifuged for 10
ST:STUDY_SUMMARY                 	min at 17,000 g. Supernatants were collected and evaporated using a Nitrogen
ST:STUDY_SUMMARY                 	evaporator (Organomation). Evaporated extracts were stored at -80°C. Dried
ST:STUDY_SUMMARY                 	metabolites were re-suspended in 100 µL (1:1) dH20, ACN solution. 10% of these
ST:STUDY_SUMMARY                 	suspensions were injected per analysis. Samples were run on a Vanquish (Thermo
ST:STUDY_SUMMARY                 	Scientific) UHPLC system with mobile phase A (20 mM ammonium carbonate, pH 9.7)
ST:STUDY_SUMMARY                 	and mobile phase B (100% ACN) at a flow rate of 150 µL/min on a SeQuant
ST:STUDY_SUMMARY                 	ZIC-pHILIC Polymeric column (2.1 × 150 mm x 5 μm, EMD Millipore) at 35°C.
ST:STUDY_SUMMARY                 	Separation is achieved with a linear gradient from 20% A to 80% A in 20 min
ST:STUDY_SUMMARY                 	followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A
ST:STUDY_SUMMARY                 	is then held from 20.5 min to 28 min. The UHPLC is coupled to a Q-Exactive
ST:STUDY_SUMMARY                 	(Thermo Scientific) mass analyzer running in polarity switching mode at 3.2 kV
ST:STUDY_SUMMARY                 	with an MS1 resolution of 70,000.
ST:INSTITUTE                     	University of California, Los Angeles
ST:LAST_NAME                     	Li
ST:FIRST_NAME                    	Joey
ST:ADDRESS                       	615 Charles E Young Dr S, Los Angeles CA 90095
ST:EMAIL                         	joeyhli@g.ucla.edu
ST:PHONE                         	4086213407
#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           	-	Human_Naive_1	Sample source:Human PBMC | Stimulation:Naïve	RAW_FILE_NAME(Raw file name)=human-NK-cells-day0-113-F__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Human_Naive_2	Sample source:Human PBMC | Stimulation:Naïve	RAW_FILE_NAME(Raw file name)=human-NK-cells-day0-114-F__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Human_Naive_3	Sample source:Human PBMC | Stimulation:Naïve	RAW_FILE_NAME(Raw file name)=human-NK-cells-day0-115-M__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Human_IL-215_1	Sample source:Human PBMC | Stimulation:3 day IL-2/15	RAW_FILE_NAME(Raw file name)=human-NK-cells-day3-113-F__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Human_IL-215_2	Sample source:Human PBMC | Stimulation:3 day IL-2/15	RAW_FILE_NAME(Raw file name)=human-NK-cells-day3-114-F__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Human_IL-215_3	Sample source:Human PBMC | Stimulation:3 day IL-2/15	RAW_FILE_NAME(Raw file name)=human-NK-cells-day3-115-M__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Mouse_Naive_1	Sample source:Mouse spleen | Stimulation:Naïve	RAW_FILE_NAME(Raw file name)=mouse-naïve-02__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Mouse_Naive_2	Sample source:Mouse spleen | Stimulation:Naïve	RAW_FILE_NAME(Raw file name)=mouse-naïve-03__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Mouse_Naive_3	Sample source:Mouse spleen | Stimulation:Naïve	RAW_FILE_NAME(Raw file name)=mouse-naïve-04__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Mouse_IL-215_1	Sample source:Mouse spleen | Stimulation:3 day IL-2/15	RAW_FILE_NAME(Raw file name)=mouse-IL-2-15-02__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Mouse_IL-215_2	Sample source:Mouse spleen | Stimulation:3 day IL-2/15	RAW_FILE_NAME(Raw file name)=mouse-IL-2-15-03__.RAW
SUBJECT_SAMPLE_FACTORS           	-	Mouse_IL-215_3	Sample source:Mouse spleen | Stimulation:3 day IL-2/15	RAW_FILE_NAME(Raw file name)=mouse-IL-2-15-04__.RAW
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were rinsed with ice-cold 150 mM NH4AcO, pH 7.3, resuspended in 80%
CO:COLLECTION_SUMMARY            	ice-cold MeOH extraction buffer, and centrifuged for 10 min at 17,000 g.
CO:COLLECTION_SUMMARY            	Supernatants were collected and evaporated using a Nitrogen evaporator
CO:COLLECTION_SUMMARY            	(Organomation). Evaporated extracts were stored at -80°C.
CO:SAMPLE_TYPE                   	Mononuclear cells
#TREATMENT
TR:TREATMENT_SUMMARY             	For cytokine stimulation, 2 x 104 mouse NK cells were cultured for 4 h at 37°C
TR:TREATMENT_SUMMARY             	in CR-10 medium (RPMI 1640 + 10% FBS, 1% L-glutamine, 1% 200 mM sodium pyruvate,
TR:TREATMENT_SUMMARY             	1% MEM-NEAA, 1% penicillin-streptomycin, 0.5% sodium bicarbonate, and 0.01% 55
TR:TREATMENT_SUMMARY             	mM 2-mercaptoethanol), recombinant mouse IL-2 (100 IU/mL), and recombinant mouse
TR:TREATMENT_SUMMARY             	IL-15 (50 ng/mL, PeproTech). Isolated human NK cells were cultured in 24-well
TR:TREATMENT_SUMMARY             	G-Rex plates (Wilson Wolf) in NK MACS medium (Miltenyi Biotech) supplemented
TR:TREATMENT_SUMMARY             	with recombinant human IL-2 (100 IU/mL, PeproTech) and recombinant human IL-15
TR:TREATMENT_SUMMARY             	(20 ng/mL, Peprotech).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Dried metabolites were re-suspended in 100 µL (1:1) dH20, ACN solution.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
CH:SOLVENT_A                     	ammonium carbonate
CH:SOLVENT_B                     	ACN
CH:FLOW_GRADIENT                 	0-20 min: 20%-80%; 20-20.5 min: 80%-20%; 20.5-28 min: 20%
CH:FLOW_RATE                     	150 uL/min
CH:COLUMN_TEMPERATURE            	35
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Metabolites were identified via exact mass (MS1), retention time, and
MS:MS_COMMENTS                   	fragmentation patterns (MS2) at normalized collision energy (NCE) 35. Relative
MS:MS_COMMENTS                   	quantification was performed via area under the curve (AUC) integration of MS1
MS:MS_COMMENTS                   	ion chromatograms with the MZmine 2 software package.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	normalized intensity
MS_METABOLITE_DATA_START
Samples	Human_Naive_1	Human_Naive_2	Human_Naive_3	Human_IL-215_1	Human_IL-215_2	Human_IL-215_3	Mouse_Naive_1	Mouse_Naive_2	Mouse_Naive_3	Mouse_IL-215_1	Mouse_IL-215_2	Mouse_IL-215_3
Factors	Sample source:Human PBMC | Stimulation:Naïve	Sample source:Human PBMC | Stimulation:Naïve	Sample source:Human PBMC | Stimulation:Naïve	Sample source:Human PBMC | Stimulation:3 day IL-2/15	Sample source:Human PBMC | Stimulation:3 day IL-2/15	Sample source:Human PBMC | Stimulation:3 day IL-2/15	Sample source:Mouse spleen | Stimulation:Naïve	Sample source:Mouse spleen | Stimulation:Naïve	Sample source:Mouse spleen | Stimulation:Naïve	Sample source:Mouse spleen | Stimulation:3 day IL-2/15	Sample source:Mouse spleen | Stimulation:3 day IL-2/15	Sample source:Mouse spleen | Stimulation:3 day IL-2/15
acetyl-carnitine	0.000000631	0.00000183	0.00000126	0.00000024	0.000000441	0.000000464	0.000000722	0.000000793	0.000000557	0.000000372	0.000000436	0.000000411
alanine	5.05E-08	5.13E-08	0.000000032	0.00000115	0.00000191	0.00000118	1.72E-08	3.09E-08	2.08E-08	9.69E-08	0.000000217	8.63E-08
arginine	0.00000126	0.0000013	0.000000697	0.00000411	0.00000752	0.00000434	1.45E-08	3.71E-08	1.73E-08	0.00000035	0.000000471	0.000000238
asparagine	0.000000131	9.62E-08	7.12E-08	2.49E-08	3.47E-08	2.86E-08	1.88E-08	2.06E-08	3.07E-08	0.000000653	0.00000183	0.000000505
betaine	0.000000365	0.000000231	0.000000109	0.000000148	0.000000185	9.29E-08	0.000000204	0.000000455	0.000000187	0.000000625	0.000000909	0.000000562
carnitine	0.0000001	0.000000266	0.000000214	0.000000159	0.000000314	0.000000237	0.000000124	0.000000451	0.000000639	0.000000347	0.00000043	0.000000452
choline	0.00000151	0.00000102	0.000000784	0.00000626	0.00000793	0.00000569	0.000000391	0.000000458	0.00000037	0.000000398	0.000000447	0.000000392
citrulline	4.81E-08	5.04E-08	2.47E-08	1.31E-08	2.34E-08	2.87E-08	8.83E-09	1.31E-08	2.34E-08	1.48E-08	1.95E-08	1.15E-08
creatine	0.00000085	0.000000508	0.000000354	0.000000314	0.00000028	0.000000277	1.17E-08	1.78E-08	1.42E-08	0.000000067	0.00000017	5.94E-08
creatinine	0.000000206	0.000000267	0.000000157	0.00000012	0.000000253	0.000000148	4.84E-09	2.65E-08	9.58E-09	4.97E-08	6.93E-08	4.24E-08
glycerophosphocholine	0.000000402	0.000000292	0.000000236	0.00000944	0.00000653	0.0000095	0.000000024	2.86E-08	3.65E-08	0.000000251	0.00000039	0.000000184
glycine	3.91E-09	2.71E-09	2.11E-09	6.34E-09	0.00000001	7.6E-09	3.47E-09	4.57E-09	9.27E-09	2.87E-08	0.00000006	2.98E-08
leucine / isoleucine	9.21E-08	7.78E-08	4.83E-08	0.00000118	0.00000225	0.00000133	2.65E-08	0.000000133	7.28E-08	0.000000414	0.000000761	0.000000452
lysine	3.02E-08	2.93E-08	1.98E-08	0.000000605	0.00000125	0.00000073	6.43E-09	1.27E-08	1.03E-08	1.55E-08	1.82E-08	1.06E-08
methionine	2.53E-08	3.85E-08	1.87E-08	0.000000678	0.000000736	0.000000391	4.14E-09	5.77E-09	4.13E-09	6.78E-08	7.81E-08	5.69E-08
methioninesulfoxide	3.88E-08	3.12E-08	1.88E-08	0.000000431	0.000000835	0.000000397	0	1.24E-08	3.3E-09	6.64E-09	0	9.88E-09
NAD+	1.23E-08	1.06E-08	7.02E-09	5.57E-08	4.49E-08	0.000000041	3.57E-08	3.43E-08	5.12E-08	0.000000141	0.000000305	0.000000193
nicotinamide	0.000000114	6.08E-08	6.87E-08	0.0000015	0.00000219	0.00000158	9.88E-09	1.42E-08	7.4E-09	5.42E-08	7.71E-08	5.99E-08
ornithine	8.65E-09	1.63E-08	4.57E-09	1.29E-08	1.56E-08	0.000000012	0.000000002	5.03E-09	2.47E-08	1.39E-08	2.49E-08	1.56E-08
phenylalanine	7.64E-08	8.15E-08	4.54E-08	0.00000119	0.00000231	0.00000136	0.000000012	6.17E-08	3.41E-08	9.82E-08	0.000000203	0.00000012
phosphocholine	0.00000778	0.00000749	0.00000576	0.0000115	0.0000101	0.0000112	0.000000444	0.00000038	0.000000499	0.00000171	0.00000228	0.00000101
proline	0.00000181	0.00000204	0.0000014	0.00000326	0.00000519	0.00000343	8.22E-09	1.34E-08	1.46E-08	0.000000197	0.000000567	0.000000265
propionyl-carnitine	1.99E-09	4.06E-09	4.02E-09	1.98E-09	3.91E-09	4.04E-09	1.38E-08	2.16E-08	2.69E-08	6.24E-09	0	8.82E-09
sarcosine	3.28E-09	6.86E-09	1.78E-09	1.47E-08	2.79E-08	2.49E-08	7.47E-10	9.57E-10	1.58E-09	4.86E-09	4.56E-09	3.12E-09
serine	2.77E-08	2.16E-08	1.58E-08	5.53E-08	0.000000107	6.85E-08	1.92E-08	0.000000021	9.77E-08	0.000000169	0.000000416	0.000000126
threonine	0.000000106	0.000000107	0.000000082	0.000000704	0.00000136	0.000000768	2.03E-08	2.39E-08	4.63E-08	0.000000103	0.000000208	0.000000091
valine	5.72E-08	0.000000058	3.74E-08	0.000000449	0.000000826	0.000000531	9.62E-09	3.07E-08	2.86E-08	5.37E-08	8.46E-08	0.000000051
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
acetyl-carnitine
alanine
arginine
asparagine
betaine
carnitine
choline
citrulline
creatine
creatinine
glycerophosphocholine
glycine
leucine / isoleucine
lysine
methionine
methioninesulfoxide
NAD+
nicotinamide
ornithine
phenylalanine
phosphocholine
proline
propionyl-carnitine
sarcosine
serine
threonine
valine
METABOLITES_END
#END