#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