#METABOLOMICS WORKBENCH hossein_maleki_20200130_134026_mwtab.txt DATATRACK_ID:1914 STUDY_ID:ST001314 ANALYSIS_ID:AN002188 PROJECT_ID:PR000892
VERSION             	1
CREATED_ON             	February 3, 2020, 2:24 pm
#PROJECT
PR:PROJECT_TITLE                 	Metabolomic Profiles of Pancreatic β-Cells and Islets Exposed to Arsenic
PR:PROJECT_SUMMARY               	Type-2 diabetes (T2D) is a complex metabolic disorder that affects hundreds of
PR:PROJECT_SUMMARY               	millions of people world-wide and is a growing public health concern. Despite
PR:PROJECT_SUMMARY               	recent advances in T2D research, the etiology of this disease and the mechanisms
PR:PROJECT_SUMMARY               	underlying the metabolic defects remain poorly understood. While obesity is
PR:PROJECT_SUMMARY               	thought to be the main cause for the rising prevalence of T2D, obesity alone
PR:PROJECT_SUMMARY               	cannot explain differences in the trends of T2D among different geographical
PR:PROJECT_SUMMARY               	regions and populations. Growing evidence suggests that environmental exposures
PR:PROJECT_SUMMARY               	to toxic and diabetogenic substances must play important roles. Inorganic
PR:PROJECT_SUMMARY               	arsenic (iAs) is a naturally occurring toxic metalloid. Hundreds of millions of
PR:PROJECT_SUMMARY               	people worldwide are exposed to unsafe levels of iAs in drinking water and food.
PR:PROJECT_SUMMARY               	iAs is a potent carcinogen, but iAs exposure has also been linked to increase
PR:PROJECT_SUMMARY               	risk of T2D. While the link between iAs exposure and T2D is well-established,
PR:PROJECT_SUMMARY               	the mechanisms underlying the diabetogenic effects of iAs exposure remain
PR:PROJECT_SUMMARY               	unclear. Results of our previously published and ongoing studies suggest that
PR:PROJECT_SUMMARY               	pancreatic β-cells are a primary target for iAs and its metabolites and that
PR:PROJECT_SUMMARY               	impaired insulin secretion by β-cells is the mechanism by which iAs exposure
PR:PROJECT_SUMMARY               	leads to diabetes. The proposed project will use metabolomics to identify
PR:PROJECT_SUMMARY               	metabolic pathways in β-cells and pancreatic islets that are targeted by iAs
PR:PROJECT_SUMMARY               	and its metabolites, monomethyl-As (MAs) and dimethyl-As (DMAs). The
PR:PROJECT_SUMMARY               	metabolomics data combined with results of our ongoing mechanistic studies will
PR:PROJECT_SUMMARY               	provide a comprehensive picture of the metabolic dysfunction leading to the
PR:PROJECT_SUMMARY               	development of diabetes in individuals exposed to iAs and of the molecular
PR:PROJECT_SUMMARY               	mechanisms that underlie this dysfunction. Identifying the affected pathways and
PR:PROJECT_SUMMARY               	mechanisms will ultimately help to improve strategies for prevention and/or
PR:PROJECT_SUMMARY               	treatment of T2D associated with chronic exposure to iAs.
PR:INSTITUTE                     	University of North Carolina at Chapel Hill
PR:LAST_NAME                     	Styblo
PR:FIRST_NAME                    	Miroslav
PR:ADDRESS                       	Departmnet of Nutrition, CB# 7461, Chapel Hill, NC 27599-7461
PR:EMAIL                         	miroslav_styblo@med.unc.edu
PR:PHONE                         	(919) 966-5721
#STUDY
ST:STUDY_TITLE                   	Metabolomic Profiles of Pancreatic β-Cells and Islets Exposed to Arsenic,
ST:STUDY_TITLE                   	Islets (part-II)
ST:STUDY_SUMMARY                 	Type-2 diabetes (T2D) is a complex metabolic disorder that affects hundreds of
ST:STUDY_SUMMARY                 	millions of people world-wide and is a growing public health concern. Despite
ST:STUDY_SUMMARY                 	recent advances in T2D research, the etiology of this disease and the mechanisms
ST:STUDY_SUMMARY                 	underlying the metabolic defects remain poorly understood. While obesity is
ST:STUDY_SUMMARY                 	thought to be the main cause for the rising prevalence of T2D, obesity alone
ST:STUDY_SUMMARY                 	cannot explain differences in the trends of T2D among different geographical
ST:STUDY_SUMMARY                 	regions and populations. Growing evidence suggests that environmental exposures
ST:STUDY_SUMMARY                 	to toxic and diabetogenic substances must play important roles. Inorganic
ST:STUDY_SUMMARY                 	arsenic (iAs) is a naturally occurring toxic metalloid. Hundreds of millions of
ST:STUDY_SUMMARY                 	people worldwide are exposed to unsafe levels of iAs in drinking water and food.
ST:STUDY_SUMMARY                 	iAs is a potent carcinogen, but iAs exposure has also been linked to increase
ST:STUDY_SUMMARY                 	risk of T2D. While the link between iAs exposure and T2D is well-established,
ST:STUDY_SUMMARY                 	the mechanisms underlying the diabetogenic effects of iAs exposure remain
ST:STUDY_SUMMARY                 	unclear. Results of our previously published and ongoing studies suggest that
ST:STUDY_SUMMARY                 	pancreatic islets are a primary target for iAs and its metabolites and that
ST:STUDY_SUMMARY                 	impaired insulin secretion by islets is the mechanism by which iAs exposure
ST:STUDY_SUMMARY                 	leads to diabetes. The proposed project will use metabolomics to identify
ST:STUDY_SUMMARY                 	metabolic pathways in β-cells that are targeted by iAs and its metabolites,
ST:STUDY_SUMMARY                 	monomethyl-As (MAs) and dimethyl-As (DMAs). The metabolomics data combined with
ST:STUDY_SUMMARY                 	results of our ongoing mechanistic studies will provide a comprehensive picture
ST:STUDY_SUMMARY                 	of the metabolic dysfunction leading to the development of diabetes in
ST:STUDY_SUMMARY                 	individuals exposed to iAs and of the molecular mechanisms that underlie this
ST:STUDY_SUMMARY                 	dysfunction. Identifying the affected pathways and mechanisms will ultimately
ST:STUDY_SUMMARY                 	help to improve strategies for prevention and/or treatment of T2D associated
ST:STUDY_SUMMARY                 	with chronic exposure to iAs.
ST:INSTITUTE                     	University of North Carolina at Chapel Hill
ST:LAST_NAME                     	Sumner
ST:FIRST_NAME                    	Susan
ST:ADDRESS                       	500 Laureate Way, Kannapolis, NC 28081
ST:EMAIL                         	Susan_sumner@unc.edu
ST:PHONE                         	(919)6224456
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENDER                        	Male
SU:CELL_PRIMARY_IMMORTALIZED     	Islets isolated from pancreata of adult C57BL/J6 male mice
SU:CELL_COUNTS                   	100 islets
#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           	-	CI_29	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_29.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_22	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_22.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_15	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_15.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_33	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_33.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_14	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_14.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_20	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_20.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_17	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_17.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_19	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_19.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_36	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_36.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_21	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_21.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_8	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_8.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_39	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_39.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_38	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_38.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_3	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_3.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_40	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_40.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_27	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_27.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_6	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_6.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_9	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_9.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_18	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_18.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_35	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_35.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_23	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_23.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_25	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_25.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_32	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_32.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_13	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_13.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_37	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_37.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_26	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_26.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_2	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_12	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_12.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_11	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_11.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_34	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_34.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_31	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_31.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_10	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_10.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_28	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_28.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_1	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_24	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_24.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_5	Cell type:Islet | Treatment:No treatment	RAW_FILE_NAME=CI_5.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_30	Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite)	RAW_FILE_NAME=CI_30.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_16	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_16.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_7	Cell type:Islet | Treatment:monomethylated arsenic	RAW_FILE_NAME=CI_7.RAW
SUBJECT_SAMPLE_FACTORS           	-	CI_4	Cell type:Islet | Treatment:dimethylated arsenic	RAW_FILE_NAME=CI_4.RAW
SUBJECT_SAMPLE_FACTORS           	-	SP_CL_1_1	Cell type:Islet sample pool | Treatment:Islet sample pool	RAW_FILE_NAME=SP_CL_1_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	SP_CL_1_2	Cell type:Islet sample pool | Treatment:Islet sample pool	RAW_FILE_NAME=SP_CL_1_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	SP_CL_2_1	Cell type:Islet sample pool | Treatment:Islet sample pool	RAW_FILE_NAME=SP_CL_2_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	SP_CL_2_2	Cell type:Islet sample pool | Treatment:Islet sample pool	RAW_FILE_NAME=SP_CL_2_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	SP_CL_3_1	Cell type:Islet sample pool | Treatment:Islet sample pool	RAW_FILE_NAME=SP_CL_3_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	SP_CL_3_2	Cell type:Islet sample pool | Treatment:Islet sample pool	RAW_FILE_NAME=SP_CL_3_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	SP_CL_4	Cell type:Islet sample pool | Treatment:Islet sample pool	RAW_FILE_NAME=SP_CL_4.RAW
#COLLECTION
CO:COLLECTION_SUMMARY            	Pancreatic islets were isolated from adult male C57BL/6 mice (Charles River
CO:COLLECTION_SUMMARY            	Laboratories, Wilmington, MA). All procedures involving mice were approved by
CO:COLLECTION_SUMMARY            	the University of North Carolina Institutional Animal and Use Committee. Mice
CO:COLLECTION_SUMMARY            	were sacrificed by cervical dislocation and pancreas was infused in situwith
CO:COLLECTION_SUMMARY            	collagenase P (1 mg/ml, Roche Diagnostics Corp., Indianapolis, IN) via the
CO:COLLECTION_SUMMARY            	common bile duct. Pancreas was then removed and digested in the collagenase
CO:COLLECTION_SUMMARY            	solution for 12 min at 37 °C. The digestate was washed and islets were purified
CO:COLLECTION_SUMMARY            	by centrifugation in a gradient of Ficoll PM 400 (GE Healthcare, Uppsala,
CO:COLLECTION_SUMMARY            	Sweden) (Szot et al., 2007).The isolated islets were cultivated overnight at 37
CO:COLLECTION_SUMMARY            	°C with 5% CO2 in RPMI 1640 medium (Mediatech, Manassas, VA) with 10% fetal
CO:COLLECTION_SUMMARY            	bovine serum, 10 mM Hepes, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100
CO:COLLECTION_SUMMARY            	μg/ml streptomycin (all from Sigma-Aldrich, St. Louis, MO). The same medium was
CO:COLLECTION_SUMMARY            	used in experiments in which the islets were exposed to iAsIII (sodium arsenite;
CO:COLLECTION_SUMMARY            	Sigma-Aldrich), MAsIII(methylarsine oxide) or DMAsIII (iododimethylarsine).
CO:SAMPLE_TYPE                   	Pancreas
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	The isolated islets were incubated overnight at 37°C with 5% CO2 at 6-well
TR:TREATMENT_SUMMARY             	plates in RPMI 1640 medium with 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium
TR:TREATMENT_SUMMARY             	pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco). The
TR:TREATMENT_SUMMARY             	islets were then exposed to 2 μM iAsIII, 0.25 μM MAsIII, or 0.5 μM DMAsIII
TR:TREATMENT_SUMMARY             	for 48 hours. Control islets were incubated in the culture medium without
TR:TREATMENT_SUMMARY             	arsenicals.
TR:CELL_STORAGE                  	-80 °C
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	With a randomized order, islet samples were put on dry ice before sample
SP:SAMPLEPREP_SUMMARY            	preparation. A volume of 400 uL ice cold methanol-water (80:20) was added, and
SP:SAMPLEPREP_SUMMARY            	then vortex for 5 min at 5000 rpm. All contents in the tube were transferred
SP:SAMPLEPREP_SUMMARY            	into pre-labeled MagNaLyzer tubes (with 10-15 beads inside). The tubes were put
SP:SAMPLEPREP_SUMMARY            	on bead homogenizer using quick run setting for bacterial cells (2 ml) with
SP:SAMPLEPREP_SUMMARY            	speed at 6.30 m/s for 45 sec in 1 cycle. All samples were centrifuged at 16,000
SP:SAMPLEPREP_SUMMARY            	rcf for 20 min at 4°C. A volume of 300 µl supernatant were transferred into
SP:SAMPLEPREP_SUMMARY            	pre-labeled 1.5 ml Low-bind Eppendorf tube. For quality control purpose, 32 µl
SP:SAMPLEPREP_SUMMARY            	of the supernatant from individual sample was transferred and mixed in new 2-ml
SP:SAMPLEPREP_SUMMARY            	tube to make a sample pool (SP). A volume of 300 µl SP was transferred into a
SP:SAMPLEPREP_SUMMARY            	pre-labeled 2.0 ml Low-bind Eppendorf tubes. All samples, including study
SP:SAMPLEPREP_SUMMARY            	samples and pools, were dried under speed-vac. For immediate analysis, 100 µL
SP:SAMPLEPREP_SUMMARY            	of Water-Methanol (95:5) containing 500 ng/mL L-tryphtophan-d5 was added to the
SP:SAMPLEPREP_SUMMARY            	residue, and then vortexed for 10 mins at 5000 rpm. After centrifuge at 4°C,
SP:SAMPLEPREP_SUMMARY            	16000 rcf for 4 min, the supernatant from individual sample was transferred to
SP:SAMPLEPREP_SUMMARY            	pre-labeled auto-sampler vial for LC-MS analysis.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um)
CH:FLOW_RATE                     	0.4 ml/min
CH:COLUMN_TEMPERATURE            	50
CH:SOLVENT_A                     	Water with 0.1% Formic Acid
CH:SOLVENT_B                     	Methanol with 0.1% Formic Acid
CH:COLUMN_PRESSURE               	6000-10000 psi
CH:INJECTION_TEMPERATURE         	8
CH:ANALYTICAL_TIME               	22 min
CH:CAPILLARY_VOLTAGE             	3.75 KV
CH:WEAK_WASH_SOLVENT_NAME        	10:90 Methanol:Water with 0.1% FA solution
CH:STRONG_WASH_SOLVENT_NAME      	75:25 2-Propanol: Water with 0.1% FA solution
CH:RANDOMIZATION_ORDER           	Yes
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	UNC-NRI Sumner Lab
AN:OPERATOR_NAME                 	Yuanyuan Li
AN:DETECTOR_TYPE                 	Orbitrap
AN:SOFTWARE_VERSION              	Xcalibur 4.1
AN:DATA_FORMAT                   	profile
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF hybrid Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	We used DDA mode to acquire the MS and MS/MS data. Progenesis QI was used for
MS:MS_COMMENTS                   	peak picking, alignment, and normalization.
MS:CAPILLARY_TEMPERATURE         	275 °C
MS:CAPILLARY_VOLTAGE             	3.75 KV
MS:COLLISION_ENERGY              	10-35, ramp
MS:COLLISION_GAS                 	N2
MS:DRY_GAS_FLOW                  	45
MS:DRY_GAS_TEMP                  	325°C
MS:FRAGMENTATION_METHOD          	CID
MS:IONIZATION                    	ES+
MS:MASS_ACCURACY                 	5ppm
MS:DATAFORMAT                    	Profile
MS:DESOLVATION_GAS_FLOW          	45
MS:DESOLVATION_TEMPERATURE       	325°C
MS:RESOLUTION_SETTING            	120000
MS:SCAN_RANGE_MOVERZ             	70-1050 m/z
MS:SCANNING_RANGE                	70-1050 m/z
MS:MS_RESULTS_FILE               	ST001314_AN002188_Results.txt	UNITS:Peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END