#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