#METABOLOMICS WORKBENCH amitch_20160831_104813 DATATRACK_ID:716 STUDY_ID:ST000474 ANALYSIS_ID:AN000738 PROJECT_ID:PR000362
VERSION             	1
CREATED_ON             	September 13, 2016, 2:11 pm
#PROJECT
PR:PROJECT_TITLE                 	Metabolomic testing of a putative MDH inhibitor
PR:PROJECT_SUMMARY               	Testing for metabolomic changes after treatment of leukemia cells with a
PR:PROJECT_SUMMARY               	putative MDH inhibitor
PR:INSTITUTE                     	Case Western Reserve University
PR:DEPARTMENT                    	Pathology
PR:LABORATORY                    	Foundation and Institutional funds
PR:LAST_NAME                     	Wald
PR:FIRST_NAME                    	David
PR:ADDRESS                       	2103 Cornell Rd WRB3-530 Cleveland, OH 44106
PR:EMAIL                         	dnw@case.edu
PR:PHONE                         	NA
#STUDY
ST:STUDY_TITLE                   	Metabolomic testing of a putative MDH inhibitor (targeted)
ST:STUDY_TYPE                    	drug treatment (single dose and timepoint)
ST:STUDY_SUMMARY                 	HL-60 cells will be treated for 15hr with a putative MDH inhibitor or vehicle
ST:STUDY_SUMMARY                 	(n=5 per group)
ST:INSTITUTE                     	University of Florida
ST:DEPARTMENT                    	SECIM
ST:LABORATORY                    	Case Western University
ST:LAST_NAME                     	Wald
ST:FIRST_NAME                    	David
ST:ADDRESS                       	2103 Cornell Rd WRB3-530 Cleveland, OH 44106
ST:EMAIL                         	dnw@case.edu
ST:PHONE                         	2163685668
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	20
ST:STUDY_COMMENTS                	Total subjects: 10 for global testing and 10 for targeted testing
#SUBJECT
SU:SUBJECT_TYPE                  	Human leukemia cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:CELL_STRAIN_DETAILS           	HL-60
SU:SUBJECT_COMMENTS              	immortalized
SU:CELL_COUNTS                   	10 million per sample
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	untreat	ut1	Treatment:-	
SUBJECT_SAMPLE_FACTORS           	untreat	ut2	Treatment:-	
SUBJECT_SAMPLE_FACTORS           	untreat	ut3	Treatment:-	
SUBJECT_SAMPLE_FACTORS           	untreat	ut4	Treatment:-	
SUBJECT_SAMPLE_FACTORS           	untreat	ut5	Treatment:-	
SUBJECT_SAMPLE_FACTORS           	treat	tx1	Treatment:MDH inhib	
SUBJECT_SAMPLE_FACTORS           	treat	tx2	Treatment:MDH inhib	
SUBJECT_SAMPLE_FACTORS           	treat	tx3	Treatment:MDH inhib	
SUBJECT_SAMPLE_FACTORS           	treat	tx4	Treatment:MDH inhib	
SUBJECT_SAMPLE_FACTORS           	treat	tx5	Treatment:MDH inhib	
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were centrifuged to remove growth media/drug and either flash frozen or
CO:COLLECTION_SUMMARY            	washed 1x in PBS. The cells were washed for the targeted assay but not for the
CO:COLLECTION_SUMMARY            	global assay.
CO:SAMPLE_TYPE                   	cell
CO:STORAGE_CONDITIONS            	flash frozen
CO:TISSUE_CELL_QUANTITY_TAKEN    	10 million per sample
#TREATMENT
TR:TREATMENT_SUMMARY             	Cells were treated for 15hr with 1 micromolar of drug or vehicle
TR:CELL_GROWTH_CONTAINER         	flasks
TR:CELL_GROWTH_RATE              	~24hr per division
TR:CELL_MEDIA                    	RPMI with 10% FBS
TR:CELL_ENVIR_COND               	5% CO2 incubator
TR:CELL_MEDIA_LASTCHANGED        	15 hr
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Amino Acid|Global
SP:SAMPLEPREP_PROTOCOL_FILENAME  	SB_OA_Assay.pdf;Appendix C_Protein_DNA_quant.pdf
SP:SAMPLE_SPIKING                	Appendix A - Internal Standard Prep GLCMS.pdf
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Amino acids
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
MS:INSTRUMENT_NAME               	Thermo Quantiva QQQ
CH:COLUMN_NAME                   	Waters Acquity BEH C18 (100 x 2mm, 1.7um)
CH:METHODS_FILENAME              	SB_OA_Assay.pdf
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:MS_COMMENTS                   	-
MS:INSTRUMENT_NAME               	Thermo Quantiva QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	Total nmoles (with relative protein adjustment)
MS_METABOLITE_DATA_START
Samples	ut1	ut2	ut3	ut4	ut5	tx1	tx2	tx3	tx4	tx5
Factors	Treatment:-	Treatment:-	Treatment:-	Treatment:-	Treatment:-	Treatment:MDH inhib	Treatment:MDH inhib	Treatment:MDH inhib	Treatment:MDH inhib	Treatment:MDH inhib
Lactate	41.94	46.29	36.76	40.93	39.02	21.16	16.53	15.44	18.54	14.91
Pyruvate	0.55	0.75	0.60	0.43	0.37	0.12	0.25	0.16	0.33	0.19
_3_HBA										
Succinate	1.18	1.32	0.79	1.06	1.10	0.37	0.21	0.17	0.21	0.18
Fumarate	0.33	0.29	0.36	0.47	0.49	0.21	0.22	0.17	0.20	0.18
Malate	4.91	4.77	3.88	4.70	4.71	2.15	2.02	1.66	1.97	1.70
a-KG	1.37	1.53	1.67	1.80	1.62	0.60	0.47	0.34	0.33	0.46
Citrate	3.41	1.85	3.41	3.42	3.42	1.66			1.75	1.43
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	retention index	quantified m/z	PubChem ID	KEGG ID
Lactate	3.98	196.1	107689
Pyruvate	6.68	299.1	1060
_3_HBA	4.21	210.1	441
Succinate	5.44	329.1	1110
Fumarate	5.59	327.1	444972
Malate	5.28	345.1	525
a-KG	6.59	462.2	164533
Citrate	5.95	508.25	311
METABOLITES_END
#END