#METABOLOMICS WORKBENCH ppzhang_20210427_201038 DATATRACK_ID:2605 STUDY_ID:ST001776 ANALYSIS_ID:AN002883 PROJECT_ID:PR001130 VERSION 1 CREATED_ON April 30, 2021, 5:06 am #PROJECT PR:PROJECT_TITLE Study on the Metabolic Response of HEK 293 Cells Exposed to Methylmercury PR:PROJECT_SUMMARY HEK 293 cells were treated with 7.5 μM methylmercury (HgMe) for 48 h. PR:PROJECT_SUMMARY Metabolites were extracted and subjected to LC-HRMS based metabolomics. LC-HRMS PR:PROJECT_SUMMARY data were processed by SIEVE2.2 software. Metabolites associated with HgMe PR:PROJECT_SUMMARY toxicity were screened and identified. PR:INSTITUTE University of Macau PR:LAST_NAME Zhang PR:FIRST_NAME pw PR:ADDRESS Taipa, Macau SAR, China PR:EMAIL yb47620@connect.um.edu.mo PR:PHONE 8613924251358 #STUDY ST:STUDY_TITLE Study on Metabolic Response of HEK 293 Cells Exposed to Methylmercury ST:STUDY_SUMMARY HEK 293 cells were treated with 7.5 μM methylmercury (HgMe) for 48 h. ST:STUDY_SUMMARY Metabolites were extracted and subjected to LC-HRMS based metabolomics. LC-HRMS ST:STUDY_SUMMARY data were processed by SIEVE2.2 software. Metabolites associated with HgMe ST:STUDY_SUMMARY toxicity were screened and identified. ST:INSTITUTE University of Macau ST:LAST_NAME Zhang ST:FIRST_NAME pw ST:ADDRESS Taipa, Macau SAR, China ST:EMAIL yb47620@um.edu.mo ST:PHONE 8613924251358 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Female #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 - S1R1 Treatment:Control RAW_FILE_NAME=S1R1 SUBJECT_SAMPLE_FACTORS - S1R2 Treatment:Control RAW_FILE_NAME=S1R2 SUBJECT_SAMPLE_FACTORS - S1R3 Treatment:Control RAW_FILE_NAME=S1R3 SUBJECT_SAMPLE_FACTORS - S2R1 Treatment:Control RAW_FILE_NAME=S2R1 SUBJECT_SAMPLE_FACTORS - S2R2 Treatment:Control RAW_FILE_NAME=S2R2 SUBJECT_SAMPLE_FACTORS - S2R3 Treatment:Control RAW_FILE_NAME=S2R3 SUBJECT_SAMPLE_FACTORS - S3R1 Treatment:Control RAW_FILE_NAME=S3R1 SUBJECT_SAMPLE_FACTORS - S3R2 Treatment:Control RAW_FILE_NAME=S3R2 SUBJECT_SAMPLE_FACTORS - S3R3 Treatment:Control RAW_FILE_NAME=S3R3 SUBJECT_SAMPLE_FACTORS - S4R1 Treatment:Control RAW_FILE_NAME=S4R1 SUBJECT_SAMPLE_FACTORS - S4R2 Treatment:Control RAW_FILE_NAME=S4R2 SUBJECT_SAMPLE_FACTORS - S4R3 Treatment:Control RAW_FILE_NAME=S4R3 SUBJECT_SAMPLE_FACTORS - S5R1 Treatment:Control RAW_FILE_NAME=S5R1 SUBJECT_SAMPLE_FACTORS - S5R2 Treatment:Control RAW_FILE_NAME=S5R2 SUBJECT_SAMPLE_FACTORS - S5R3 Treatment:Control RAW_FILE_NAME=S5R3 SUBJECT_SAMPLE_FACTORS - S6R1 Treatment:Control RAW_FILE_NAME=S6R1 SUBJECT_SAMPLE_FACTORS - S6R2 Treatment:Control RAW_FILE_NAME=S6R2 SUBJECT_SAMPLE_FACTORS - S6R3 Treatment:Control RAW_FILE_NAME=S6R3 SUBJECT_SAMPLE_FACTORS - S7R1 Treatment:HgMe RAW_FILE_NAME=S7R1 SUBJECT_SAMPLE_FACTORS - S7R2 Treatment:HgMe RAW_FILE_NAME=S7R2 SUBJECT_SAMPLE_FACTORS - S7R3 Treatment:HgMe RAW_FILE_NAME=S7R3 SUBJECT_SAMPLE_FACTORS - S8R1 Treatment:HgMe RAW_FILE_NAME=S8R1 SUBJECT_SAMPLE_FACTORS - S8R2 Treatment:HgMe RAW_FILE_NAME=S8R2 SUBJECT_SAMPLE_FACTORS - S8R3 Treatment:HgMe RAW_FILE_NAME=S8R3 SUBJECT_SAMPLE_FACTORS - S9R1 Treatment:HgMe RAW_FILE_NAME=S9R1 SUBJECT_SAMPLE_FACTORS - S9R2 Treatment:HgMe RAW_FILE_NAME=S9R2 SUBJECT_SAMPLE_FACTORS - S9R3 Treatment:HgMe RAW_FILE_NAME=S9R3 SUBJECT_SAMPLE_FACTORS - S10R1 Treatment:HgMe RAW_FILE_NAME=S10R1 SUBJECT_SAMPLE_FACTORS - S10R2 Treatment:HgMe RAW_FILE_NAME=S10R2 SUBJECT_SAMPLE_FACTORS - S10R3 Treatment:HgMe RAW_FILE_NAME=S10R3 SUBJECT_SAMPLE_FACTORS - S11R1 Treatment:HgMe RAW_FILE_NAME=S11R1 SUBJECT_SAMPLE_FACTORS - S11R2 Treatment:HgMe RAW_FILE_NAME=S11R2 SUBJECT_SAMPLE_FACTORS - S11R3 Treatment:HgMe RAW_FILE_NAME=S11R3 SUBJECT_SAMPLE_FACTORS - S12R1 Treatment:HgMe RAW_FILE_NAME=S12R1 SUBJECT_SAMPLE_FACTORS - S12R2 Treatment:HgMe RAW_FILE_NAME=S12R2 SUBJECT_SAMPLE_FACTORS - S12R3 Treatment:HgMe RAW_FILE_NAME=S12R3 #COLLECTION CO:COLLECTION_SUMMARY At the endpoint of the experiment, cells were washed gently with 150 mM ammonium CO:COLLECTION_SUMMARY acetate buffer and scraped quickly with a rubber-tipped cell scraper. CO:COLLECTION_SUMMARY Subsequently, 500 uL chilled 80% methanol was added and the scraped cells were CO:COLLECTION_SUMMARY sonicated using a bioruptor (Diagenode, Philadelphia, PA, USA) in 4 °C water CO:COLLECTION_SUMMARY bath during 10 cycles (30s on and 30s off). After that, the sample was placed at CO:COLLECTION_SUMMARY -20 °C for 3 h, followed by 10 min of centrifugation at 14000 g at 4 °C. The CO:COLLECTION_SUMMARY supernatant containing the metabolite was collected, dried, and stored at -80 CO:COLLECTION_SUMMARY °C until LC-HRMS analysis. CO:SAMPLE_TYPE HEK cells #TREATMENT TR:TREATMENT_SUMMARY About 1 million cells were seeded in a 25 cm2 flask. When cells reached about TR:TREATMENT_SUMMARY 50% confluence, the experimental group was changed with medium containing 7.5 uM TR:TREATMENT_SUMMARY methylmercury and the control group was changed with fresh culture medium. After TR:TREATMENT_SUMMARY 48 h treatment, cells were harvested. TR:CELL_GROWTH_CONTAINER 25 cm2 flask TR:CELL_MEDIA DMEM TR:CELL_HARVESTING 90% confluence #SAMPLEPREP SP:SAMPLEPREP_SUMMARY cells were washed gently with 150 mM ammonium acetate buffer and scraped quickly SP:SAMPLEPREP_SUMMARY with a rubber-tipped cell scraper. Subsequently, 500 uL chilled 80% methanol was SP:SAMPLEPREP_SUMMARY added and the scraped cells were sonicated using a bioruptor (Diagenode, SP:SAMPLEPREP_SUMMARY Philadelphia, PA, USA) in 4 °C water bath during 10 cycles (30s on and 30s SP:SAMPLEPREP_SUMMARY off). After that, the sample was placed at -20 °C for 3 h, followed by 10 min SP:SAMPLEPREP_SUMMARY of centrifugation at 14000 g at 4 °C. The supernatant containing the metabolite SP:SAMPLEPREP_SUMMARY was dried by speedvac and stored at -80 °C until LC-HRMS analysis.The residual SP:SAMPLEPREP_SUMMARY protein pellet was dissolved in protein extraction buffer and quantified for SP:SAMPLEPREP_SUMMARY normalization. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Sampe were reconstituted in 50% ACN. The volume was adjusted by the protein SP:SAMPLE_RESUSPENSION concentration. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The UHPLC system was equipped with a binary pump, an autosampler and a column CH:CHROMATOGRAPHY_SUMMARY thermostat. The autosampler was set at 8 °C. The column oven temperature was 30 CH:CHROMATOGRAPHY_SUMMARY °C. Mobile phase A was 98% acetonitrile, 2% water and 0.1% formic acid while CH:CHROMATOGRAPHY_SUMMARY mobile phase B was 98% water, 2% acetonitrile, 30 mM ammonium formate and 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid. The mobile phase was freshly prepared before use. The optimized CH:CHROMATOGRAPHY_SUMMARY stepwise linear gradient (10% B in the first 2 min, 10% - 30% B in the next 5 CH:CHROMATOGRAPHY_SUMMARY min, followed by 30% - 60% in 3 min, and finally 60% - 90% in 5 min. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Thermo Accucore HILIC (100 x 2.1mm, 2.6um) CH:COLUMN_TEMPERATURE 25 CH:SOLVENT_A 98% acetonitrile, 2% water and 0.1% formic acid CH:SOLVENT_B 98% water, 2% acetonitrile, 30 mM ammonium formate and 0.1% formic acid CH:SAMPLE_LOOP_SIZE 10 #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 The MS acquisition parameters were as follows: spray voltage, 3.5 kV; capillary MS:MS_COMMENTS temperature, 320 °C; sheath gas flow rate, 45; auxiliary gas flow rate, 25; MS:MS_COMMENTS heater temperature 350 °C; AGC, 3×106; maximum injection time, 200 ms; mass MS:MS_COMMENTS scan range, 50–750; full MS resolution, 70,000 FWHM at m/z 200; spectrum data MS:MS_COMMENTS type, profile. The raw LC-HRMS files for the same study were processed in one MS:MS_COMMENTS batch using the label-free differential analysis software (SIEVE 2.2, Thermo MS:MS_COMMENTS Scientific), in which the ChromAlign algorithm was used. The key parameters for MS:MS_COMMENTS the feature extraction were as follows: signal to background noise,>3; Mzstep MS:MS_COMMENTS accuracy, 10 ppm, minimum peak intensity 200,000; minimum peak scan points, 5; MS:MS_COMMENTS minimum isotopes,1. MS:MS_RESULTS_FILE ST001776_AN002883_Results.txt UNITS:arbitray unit Has m/z:Yes Has RT:Yes RT units:Minutes #END