#METABOLOMICS WORKBENCH J_aldana11_20230608_070343 DATATRACK_ID:4071 STUDY_ID:ST002734 ANALYSIS_ID:AN004432 PROJECT_ID:PR001698 VERSION 1 CREATED_ON June 8, 2023, 8:29 pm #PROJECT PR:PROJECT_TITLE Integrative multi-omics analysis of oncogenic EZH2 mutants: from epigenetic PR:PROJECT_TITLE reprogramming to molecular signatures PR:PROJECT_SUMMARY Metabolic study of Enhancer of zeste homolog 2 (EZH2) isogenic mutants with PR:PROJECT_SUMMARY gain-of-function (GOF) and loss-of-function (LOF) enzymatic activity in HEK-293T PR:PROJECT_SUMMARY cell lines. PR:INSTITUTE The Ohio State University PR:DEPARTMENT Chemistry and Biochemistry PR:LAST_NAME Aldana PR:FIRST_NAME Julian PR:ADDRESS 460 W 12th Ave, Columbus, OH 43210 PR:EMAIL aldanaaroca.1@osu.edu PR:PHONE +1 614-292-6136 #STUDY ST:STUDY_TITLE Integrative multi-omics analysis of oncogenic EZH2 mutants: from epigenetic ST:STUDY_TITLE reprogramming to molecular signatures ST:STUDY_SUMMARY Metabolic study of Enhancer of zeste homolog 2 (EZH2) isogenic mutants with ST:STUDY_SUMMARY gain-of-function (GOF) and loss-of-function (LOF) enzymatic activity in HEK-293T ST:STUDY_SUMMARY cell lines. ST:INSTITUTE The Ohio State University ST:DEPARTMENT Chemistry and Biochemistry ST:LAST_NAME Aldana ST:FIRST_NAME Julian ST:ADDRESS 460 W 12th Ave, Columbus, OH 43210 ST:EMAIL aldanaaroca.1@osu.edu ST:PHONE +1 614-292-6136 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 12 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Female SU:CELL_BIOSOURCE_OR_SUPPLIER American Tissue Culture Collection (ATCC) SU:CELL_STRAIN_DETAILS HEK-293T SU:CELL_PASSAGE_NUMBER 2 #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 - A677G_1 Factor:A677G-mutant Ionization mode=ESI (+); RAW_FILE_NAME=A677G_1.mzML SUBJECT_SAMPLE_FACTORS - A677G_2 Factor:A677G-mutant Ionization mode=ESI (+); RAW_FILE_NAME=A677G_2.mzML SUBJECT_SAMPLE_FACTORS - A677G_3 Factor:A677G-mutant Ionization mode=ESI (+); RAW_FILE_NAME=A677G_3.mzML SUBJECT_SAMPLE_FACTORS - DM_1 Factor:F667I/H689A-mutant Ionization mode=ESI (+); RAW_FILE_NAME=DM_1.mzML SUBJECT_SAMPLE_FACTORS - DM_2 Factor:F667I/H689A-mutant Ionization mode=ESI (+); RAW_FILE_NAME=DM_2.mzML SUBJECT_SAMPLE_FACTORS - DM_3 Factor:F667I/H689A-mutant Ionization mode=ESI (+); RAW_FILE_NAME=DM_3.mzML SUBJECT_SAMPLE_FACTORS - Y641F_1 Factor:Y641F-mutant Ionization mode=ESI (+); RAW_FILE_NAME=Y641F_1.mzML SUBJECT_SAMPLE_FACTORS - Y641F_2 Factor:Y641F-mutant Ionization mode=ESI (+); RAW_FILE_NAME=Y641F_2.mzML SUBJECT_SAMPLE_FACTORS - Y641F_3 Factor:Y641F-mutant Ionization mode=ESI (+); RAW_FILE_NAME=Y641F_3.mzML SUBJECT_SAMPLE_FACTORS - WT_1 Factor:Wild-type Ionization mode=ESI (+); RAW_FILE_NAME=WT_1.mzML SUBJECT_SAMPLE_FACTORS - WT_2 Factor:Wild-type Ionization mode=ESI (+); RAW_FILE_NAME=WT_2.mzML SUBJECT_SAMPLE_FACTORS - WT_3 Factor:Wild-type Ionization mode=ESI (+); RAW_FILE_NAME=WT_3.mzML SUBJECT_SAMPLE_FACTORS - QC_0 Factor:Quality control Ionization mode=ESI (+); RAW_FILE_NAME=QC_0.mzML SUBJECT_SAMPLE_FACTORS - QC_1 Factor:Quality control Ionization mode=ESI (+); RAW_FILE_NAME=QC_1.mzML SUBJECT_SAMPLE_FACTORS - QC_2 Factor:Quality control Ionization mode=ESI (+); RAW_FILE_NAME=QC_2.mzML SUBJECT_SAMPLE_FACTORS - QC_3 Factor:Quality control Ionization mode=ESI (+); RAW_FILE_NAME=QC_3.mzML SUBJECT_SAMPLE_FACTORS - QC_4 Factor:Quality control Ionization mode=ESI (+); RAW_FILE_NAME=QC_4.mzML SUBJECT_SAMPLE_FACTORS - A677G_1_neg Factor:A677G-mutant Ionization mode=ESI (-); RAW_FILE_NAME=A677G_1_neg.mzML SUBJECT_SAMPLE_FACTORS - A677G_2_neg Factor:A677G-mutant Ionization mode=ESI (-); RAW_FILE_NAME=A677G_2_neg.mzML SUBJECT_SAMPLE_FACTORS - A677G_3_neg Factor:A677G-mutant Ionization mode=ESI (-); RAW_FILE_NAME=A677G_3_neg.mzML SUBJECT_SAMPLE_FACTORS - DM_1_neg Factor:F667I/H689A-mutant Ionization mode=ESI (-); RAW_FILE_NAME=DM_1_neg.mzML SUBJECT_SAMPLE_FACTORS - DM_2_neg Factor:F667I/H689A-mutant Ionization mode=ESI (-); RAW_FILE_NAME=DM_2_neg.mzML SUBJECT_SAMPLE_FACTORS - DM_3_neg Factor:F667I/H689A-mutant Ionization mode=ESI (-); RAW_FILE_NAME=DM_3_neg.mzML SUBJECT_SAMPLE_FACTORS - Y641F_1_neg Factor:Y641F-mutant Ionization mode=ESI (-); RAW_FILE_NAME=Y641F_1_neg.mzML SUBJECT_SAMPLE_FACTORS - Y641F_2_neg Factor:Y641F-mutant Ionization mode=ESI (-); RAW_FILE_NAME=Y641F_2_neg.mzML SUBJECT_SAMPLE_FACTORS - Y641F_3_neg Factor:Y641F-mutant Ionization mode=ESI (-); RAW_FILE_NAME=Y641F_3_neg.mzML SUBJECT_SAMPLE_FACTORS - WT_1_neg Factor:Wild-type Ionization mode=ESI (-); RAW_FILE_NAME=WT_1_neg.mzML SUBJECT_SAMPLE_FACTORS - WT_2_neg Factor:Wild-type Ionization mode=ESI (-); RAW_FILE_NAME=WT_2_neg.mzML SUBJECT_SAMPLE_FACTORS - WT_3_neg Factor:Wild-type Ionization mode=ESI (-); RAW_FILE_NAME=WT_3_neg.mzML SUBJECT_SAMPLE_FACTORS - QC_0_neg Factor:Quality control Ionization mode=ESI (-); RAW_FILE_NAME=QC_0_neg.mzML SUBJECT_SAMPLE_FACTORS - QC_1_neg Factor:Quality control Ionization mode=ESI (-); RAW_FILE_NAME=QC_1_neg.mzML SUBJECT_SAMPLE_FACTORS - QC_2_neg Factor:Quality control Ionization mode=ESI (-); RAW_FILE_NAME=QC_2_neg.mzML SUBJECT_SAMPLE_FACTORS - QC_3_neg Factor:Quality control Ionization mode=ESI (-); RAW_FILE_NAME=QC_3_neg.mzML SUBJECT_SAMPLE_FACTORS - QC_4_neg Factor:Quality control Ionization mode=ESI (-); RAW_FILE_NAME=QC_4_neg.mzML #COLLECTION CO:COLLECTION_SUMMARY HEK-293T cells were acquired from ATCC CRL-1573 CO:SAMPLE_TYPE HEK cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY HEK-293T cells at 80% confluency were infected in the presence of polybrene for TR:TREATMENT_SUMMARY 16 h in DMEM with lentivirus constructs containing either wild-type (WT) EZH2, TR:TREATMENT_SUMMARY A677G-mutant (A677G) EZH2, Y641F-mutant (Y641F) EZH2, or H689A/F667I-mutant (DM) TR:TREATMENT_SUMMARY EZH2. Infected cells were selected for resistance to blasticidin (10 μg/mL) for TR:TREATMENT_SUMMARY 10 days to ensure monoclonal populations. TR:CELL_MEDIA DMEM supplemented with 10%FBS and 1% Pen/Strep TR:CELL_PCT_CONFLUENCE 80 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Approximately 10 million cells per sample were washed with PBS at room SP:SAMPLEPREP_SUMMARY tem-perature and harvested with a mixture of ice-cold methanol:water (80:20). SP:SAMPLEPREP_SUMMARY Cell lysis was carried out by four freeze-and-thaw cycles with liquid nitrogen SP:SAMPLEPREP_SUMMARY and dry-ice with vortexing between cycles. Supernatant was then dried on SP:SAMPLEPREP_SUMMARY SpeedVac concentrator and samples stored at -80ºC. Dried extracts were SP:SAMPLEPREP_SUMMARY resuspended in a mixture of Acetonitrile:Water (25:75). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 6545 CH:COLUMN_NAME Phenomenex Kinetex HILIC (100 X 2.1mm, 2.6um) CH:SOLVENT_A 100% water; 0.1 % formic acid; 10 mM ammonium formate CH:SOLVENT_B 100% acetonitrile; 0.1 % formic acid;10 mM ammonium formate CH:FLOW_GRADIENT 98–55% B in 45 min; 55% B during 4 min; 55–98% in 2 min; and column CH:FLOW_GRADIENT re-equilibration for 15 min CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS OpenMS (v2.6.0) was employed for feature detection, alignment, assembly, and MS:MS_COMMENTS de-convolution. MS:MS_RESULTS_FILE ST002734_AN004432_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END