#METABOLOMICS WORKBENCH chunhongyan_20210811_133818 DATATRACK_ID:2796 STUDY_ID:ST001909 ANALYSIS_ID:AN003106 PROJECT_ID:PR001203
VERSION             	1
CREATED_ON             	August 18, 2021, 9:46 am
#PROJECT
PR:PROJECT_TITLE                 	ATF3 regulation of serine metabolism
PR:PROJECT_TYPE                  	Isotope Tracing, GC-MS
PR:PROJECT_SUMMARY               	ATF3 is a common stress sensor, and its expression can be induced by serine
PR:PROJECT_SUMMARY               	deprivation. The goal of this project is to determine whether ATF3 regulates
PR:PROJECT_SUMMARY               	serine biosynthesis. ATF3-wildtype and –knockout cells are cultured in
PR:PROJECT_SUMMARY               	complete or serine-free medium supplemented with 13C-6-glucose for 24 h for
PR:PROJECT_SUMMARY               	stable isotope tracing. The results show that ATF3 appears to promote serine
PR:PROJECT_SUMMARY               	biosynthesis.
PR:INSTITUTE                     	Augusta University
PR:DEPARTMENT                    	Georgia Cancer Center
PR:LAST_NAME                     	Yan
PR:FIRST_NAME                    	Chunhong
PR:ADDRESS                       	1410 Laney Walker Blvd, Augusta, GA, 30912, USA
PR:EMAIL                         	cyan@augusta.edu
PR:PHONE                         	7067210099
PR:FUNDING_SOURCE                	NIH/NCI R01CA240933, R01CA190429, R01CA236890
#STUDY
ST:STUDY_TITLE                   	ATF3 regulation of serine metabolism
ST:STUDY_SUMMARY                 	ATF3 is a common stress sensor, and its expression can be induced by serine
ST:STUDY_SUMMARY                 	deprivation. The goal of this project is to determine whether ATF3 regulates
ST:STUDY_SUMMARY                 	serine biosynthesis. ATF3-wildtype and –knockout cells are cultured in
ST:STUDY_SUMMARY                 	complete or serine-free medium supplemented with 13C-6-glucose for 24 h for
ST:STUDY_SUMMARY                 	stable isotope tracing. The results show that ATF3 appears to promote serine
ST:STUDY_SUMMARY                 	biosynthesis.
ST:INSTITUTE                     	Augusta University
ST:LAST_NAME                     	Yan
ST:FIRST_NAME                    	Chunhong
ST:ADDRESS                       	1410 Laney Walker Blvd, Augusta, GA, 30912, USA
ST:EMAIL                         	cyan@augusta.edu
ST:STUDY_TYPE                    	stable isotope tracing
ST:PHONE                         	7067210099
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENOTYPE_STRAIN               	ATF3(+/+) vs ATF3(-/-)
SU:CELL_STRAIN_DETAILS           	HCT116 cells engineered to knockout ATF3 expression via rAAV-mediated homologous
SU:CELL_STRAIN_DETAILS           	recombination
#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           	-	Ding_A-1_001	Genotype_Media supplement:WT-complete	RAW_FILE_NAME=210517_Ding_A-1_001_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_A-2_002	Genotype_Media supplement:WT-complete	RAW_FILE_NAME=210517_Ding_A-2_002_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_A-3_003	Genotype_Media supplement:WT-complete	RAW_FILE_NAME=210517_Ding_A-3_003_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_A-4_004	Genotype_Media supplement:WT-complete	RAW_FILE_NAME=210517_Ding_A-4_004_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_A-5_005	Genotype_Media supplement:WT-complete	RAW_FILE_NAME=210517_Ding_A-5_005_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_A-6_006	Genotype_Media supplement:WT-complete	RAW_FILE_NAME=210517_Ding_A-6_006_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_B-1_007	Genotype_Media supplement:WT-serine(-)	RAW_FILE_NAME=210517_Ding_B-1_007_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_B-2_008	Genotype_Media supplement:WT-serine(-)	RAW_FILE_NAME=210517_Ding_B-2_008_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_B-3_009	Genotype_Media supplement:WT-serine(-)	RAW_FILE_NAME=210517_Ding_B-3_009_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_B-4_010	Genotype_Media supplement:WT-serine(-)	RAW_FILE_NAME=210517_Ding_B-4_010_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_B-5_011	Genotype_Media supplement:WT-serine(-)	RAW_FILE_NAME=210517_Ding_B-5_011_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_B-6_012	Genotype_Media supplement:WT-serine(-)	RAW_FILE_NAME=210517_Ding_B-6_012_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_C-1_013	Genotype_Media supplement:KO-complete	RAW_FILE_NAME=210517_Ding_C-1_013_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_C-2_014	Genotype_Media supplement:KO-complete	RAW_FILE_NAME=210517_Ding_C-2_014_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_C-3_015	Genotype_Media supplement:KO-complete	RAW_FILE_NAME=210517_Ding_C-3_015_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_C-4_016	Genotype_Media supplement:KO-complete	RAW_FILE_NAME=210517_Ding_C-4_016_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_C-5_017	Genotype_Media supplement:KO-complete	RAW_FILE_NAME=210517_Ding_C-5_017_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_C-6_018	Genotype_Media supplement:KO-complete	RAW_FILE_NAME=210517_Ding_C-6_018_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_D-1_019	Genotype_Media supplement:KO-serine(-)	RAW_FILE_NAME=210517_Ding_D-1_019_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_D-2_020	Genotype_Media supplement:KO-serine(-)	RAW_FILE_NAME=210517_Ding_D-2_020_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_D-3_021	Genotype_Media supplement:KO-serine(-)	RAW_FILE_NAME=210517_Ding_D-3_021_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_D-4_022	Genotype_Media supplement:KO-serine(-)	RAW_FILE_NAME=210517_Ding_D-4_022_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_D-5_023	Genotype_Media supplement:KO-serine(-)	RAW_FILE_NAME=210517_Ding_D-5_023_TBS.D
SUBJECT_SAMPLE_FACTORS           	-	Ding_D-6_024	Genotype_Media supplement:KO-serine(-)	RAW_FILE_NAME=210517_Ding_D-6_024_TBS.D
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were counted, washed with cold PBS and then flash-frozen in liquid
CO:COLLECTION_SUMMARY            	nitrogen
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	HCT116 cells (~50% confluence) were washed with PBS, and cultured in HEPES
TR:TREATMENT_SUMMARY             	buffered Krebs-Ringer solution supplemented with 25 mM [U-13C]-D-glucose, 10%
TR:TREATMENT_SUMMARY             	dialysed FBS, 2×MEM amino acids, 2× Vitamin Solution and 4 mM L-glutamine,
TR:TREATMENT_SUMMARY             	with or without 0.4 mM serine and glycine, for 24 h. Cells were washed with PBS
TR:TREATMENT_SUMMARY             	and metabolites extracted using 1 mL of acetonitrile:isopropanol:water (3:3:2,
TR:TREATMENT_SUMMARY             	v/v/v) mixture.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The extraction solvent was degassed and pre-chilled at −20°C. Samples were
SP:SAMPLEPREP_SUMMARY            	homogenized using Geno/Grinder 2010 (SPEX SamplePrep) at 1500 rpm for 30s, then
SP:SAMPLEPREP_SUMMARY            	shaken at 4°C for 5 min and centrifuged for 2 minutes at 14,000 rcf. 450 μL
SP:SAMPLEPREP_SUMMARY            	supernatant was transferred to a new tube and dried down using Centrivap cold
SP:SAMPLEPREP_SUMMARY            	trap concentrator (Labconco). 10 μL of methoxyamine hydrochloride in pyridine
SP:SAMPLEPREP_SUMMARY            	(40 mg/mL) was added to dried sample and shaken at 30°C for 1.5 hours for
SP:SAMPLEPREP_SUMMARY            	methoximation. 90 μL of N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide
SP:SAMPLEPREP_SUMMARY            	(MTBSTFA, Sigma-Aldrich) was used for tertbutylsilylation. C8–C30 fatty acid
SP:SAMPLEPREP_SUMMARY            	methyl esters (FAMEs) were added to MTBSTFA as internal standards for retention
SP:SAMPLEPREP_SUMMARY            	time correction. Samples were shaken at 37°C for 30 min for TMS or shaken at
SP:SAMPLEPREP_SUMMARY            	80°C for 30 min for TBS and then ready for injection.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Gas chromatography-Quadrupole-time of flight mass spectrometry (GC-Q-TOF MS) was
CH:CHROMATOGRAPHY_SUMMARY        	used for Stable Isotope enrichment. Rtx-5Sil MS column (30m length, 0.25 mm i.d,
CH:CHROMATOGRAPHY_SUMMARY        	0.25 μM 95% dimethyl 5% diphenyl polysiloxane film) with an additional 10 m
CH:CHROMATOGRAPHY_SUMMARY        	guard column was installed on Agilent 7890 GC (Agilent Technologies). 99.9999%
CH:CHROMATOGRAPHY_SUMMARY        	pure Helium gas was used as a mobile phase with a flow rate of 1mL/min. GC
CH:CHROMATOGRAPHY_SUMMARY        	temperature was held at 50°C for 1 min, ramped at 20°C/min to 330°C and then
CH:CHROMATOGRAPHY_SUMMARY        	held for 5 min. Electron ionization at -70eV was employed on a Agilent 7250
CH:CHROMATOGRAPHY_SUMMARY        	Quadrupole time of flight mass spectrometer (Agilent Technologies) with ion
CH:CHROMATOGRAPHY_SUMMARY        	source temperature at 250°C and detector voltage at 1850V. Mass spectra were
CH:CHROMATOGRAPHY_SUMMARY        	acquired at an acquisition rate of 5 spectra/s, with a scan range of 85-900 Da.
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890A
CH:COLUMN_NAME                   	Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	West Coast Metabolomics Center
#MS
MS:INSTRUMENT_NAME               	Agilent 7890A
MS:INSTRUMENT_TYPE               	GC-TOF
MS:MS_TYPE                       	EI
MS:ION_MODE                      	UNSPECIFIED
MS:MS_COMMENTS                   	Agilent MassHunter Quantitative Analysis software B.10.00 (Agilent Technologies)
MS:MS_COMMENTS                   	was used for post data processing.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	enrichment percentage
MS_METABOLITE_DATA_START
Samples	Ding_A-1_001	Ding_A-2_002	Ding_A-3_003	Ding_A-4_004	Ding_A-5_005	Ding_A-6_006	Ding_B-1_007	Ding_B-2_008	Ding_B-3_009	Ding_B-4_010	Ding_B-5_011	Ding_B-6_012	Ding_C-1_013	Ding_C-2_014	Ding_C-3_015	Ding_C-4_016	Ding_C-5_017	Ding_C-6_018	Ding_D-1_019	Ding_D-2_020	Ding_D-3_021	Ding_D-4_022	Ding_D-5_023	Ding_D-6_024
Factors	Genotype_Media supplement:WT-complete	Genotype_Media supplement:WT-complete	Genotype_Media supplement:WT-complete	Genotype_Media supplement:WT-complete	Genotype_Media supplement:WT-complete	Genotype_Media supplement:WT-complete	Genotype_Media supplement:WT-serine(-)	Genotype_Media supplement:WT-serine(-)	Genotype_Media supplement:WT-serine(-)	Genotype_Media supplement:WT-serine(-)	Genotype_Media supplement:WT-serine(-)	Genotype_Media supplement:WT-serine(-)	Genotype_Media supplement:KO-complete	Genotype_Media supplement:KO-complete	Genotype_Media supplement:KO-complete	Genotype_Media supplement:KO-complete	Genotype_Media supplement:KO-complete	Genotype_Media supplement:KO-complete	Genotype_Media supplement:KO-serine(-)	Genotype_Media supplement:KO-serine(-)	Genotype_Media supplement:KO-serine(-)	Genotype_Media supplement:KO-serine(-)	Genotype_Media supplement:KO-serine(-)	Genotype_Media supplement:KO-serine(-)
3-Phosphoglyceric acid	138	56	84	155	130	210	63	48	86	69	69	77	59	81	81	95	69	84	81	65	68		108	172
alanine	430	365	433	291	337	260	385	418	438	364	293	279	242	331	236	150	254	204	215	263	303	249	218	283
asparagine									27		27	18	28			21			28	35	13			
aspartic acid	39	51	41	41	30	37		33			23	24	23	25	22	30	30	43	25	22	20	29	13	20
cis-Aconitic acid		48	153	198	99			87	117	179		86	37	43	17	27	37	35	31	31	37	35	52	26
citric acid	209	252	270	269	262	273	172	264	191	188	161	227	185	151	173	132	141	140	113	149	120	107	121	153
Fumaric acid	13	15	16	15	15	18	10	11	12	11	12	13	10	10	10	11	10	11	8	8	7	8	8	9
glutamic acid	33	28	22	23	30	24	25	14	20	23	18	24	12	15	17	12	15	14	12	14	12	11	11	11
glycine				4			36	35	36	41	36	33		0	1	3	0		44	50	44	49	46	45
Isocitric acid	283	241	307	269	254	271	170	326	183	181	186	223	197	155	177	131	149	130	100	154	118	111	121	156
lactic acid	840	775	758	371	865	688	505	651	896	600	614	700	627	271	495	519	482	449	482	408	345	365	552	455
leucine						6		2	2						3			4						
malic acid	48	46	55	47	31	48	34	11	27	13	22	36	31	47	34	27	44	24	17	21	23	30	27	30
methionine		2	3	4			4		2		2	2	2			3					3			
oxoproline	1	1	3		2	1	1	2	2		2	2	3	2	2	1	1	1	2	2		1		
phenylalanine								0			0		1	1						1			1	2
proline		16	16					11		13				18	11	6	13	8	13		8			13
Pyruvate	207	344	377	342	227	129	180	220	378	160	143	199	238	566	351	122	308	131	344	142	235	196	177	300
serine	9	9	14	9	6	9	50	45	99	60	83	75	9	10	11	10	12	11	50	64	54	91	52	43
succinic acid	31	28	26	25	23	26	18	19	23	21	22	24	16	18	26	23	19	18	14	14	18	16	16	14
threonine	2	3		5				1							0			3						
tyrosine			1	1		1						1			1			1	1	3			2	
valine		0			1									1				1	1				1	1
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	meox.MSTFA derivatized formula	underivatized formula	molecular formula
3-Phosphoglyceric acid	16.441	C15H39O7PSi4	C3H7O7P
alanine	7.746	C9H23NO2Si2	C3H7NO2
asparagine	14.972	C13H32N2O3Si3	C4H8N2O3
aspartic acid	13.23	C13H31NO4Si3	C4H7NO4
cis-Aconitic acid	16.003	C15H30O6Si3	C6H6O6
citric acid	16.598	C18H40O7Si4	C6H8O7
Fumaric acid	11.043	C10H20O4Si2	C4H4O4
glutamic acid	14.41	C14H33NO4Si3	C5H9NO4
glycine	10.449	C11H29NO2Si3	C2H5NO2
Isocitric acid	16.631	C18H40O7Si4	C6H8O7
lactic acid	7.03	C9H22O3Si2	C3H6O3
leucine	9.963	C12H29NO2Si2	C6H13NO2
malic acid	12.796	C13H30O5Si3	C4H6O5
methionine	13.194	C11H27NO2SSi2	C5H11NO2S
oxoproline	13.254	C8H15NO3Si	C5H7NO3
phenylalanine	14.494	C15H27NO2Si2	C9H11NO2
proline	10.348	C11H25NO2Si2	C5H9NO2
Pyruvate	6.957	C7H15NO3Si	C3H4O3
serine	11.165	C12H31NO3Si3	C3H7NO3
succinic acid	10.559	C10H22O4Si2	C4H6O4
threonine	11.476	C13H33NO3Si3	C4H9NO3
tyrosine	17.856	C15H27NO3Si2	C9H11NO3
valine	9.202	C11H27NO2Si2	C5H11NO2
METABOLITES_END
#END