#METABOLOMICS WORKBENCH ppzhang_20190205_184541 DATATRACK_ID:1616 STUDY_ID:ST001132 ANALYSIS_ID:AN001857 PROJECT_ID:PR000757
VERSION             	1
CREATED_ON             	February 6, 2019, 12:29 pm
#PROJECT
PR:PROJECT_TITLE                 	Investigate the False Discovery in Biomarker Research Using LC-HRMS based
PR:PROJECT_TITLE                 	Untargeted Metabolomics Profiling
PR:PROJECT_SUMMARY               	Pooled human plasma was spiked separately with two different sets of 11
PR:PROJECT_SUMMARY               	metabolite standards (22 “true biomarkers”) to mimic plasma samples
PR:PROJECT_SUMMARY               	collected from diseased subjects and non-diseased subjects. Metabolite extracts
PR:PROJECT_SUMMARY               	of individual samples were subjected to biomarker discovery using LC-HRMS.
PR:INSTITUTE                     	University of Macau, Macau, China
PR:DEPARTMENT                    	Institute of Translational Medicine, Faculty of Health Sciences,
PR:LABORATORY                    	Pilot Laboratory
PR:LAST_NAME                     	ZHANG
PR:FIRST_NAME                    	pw
PR:ADDRESS                       	Taipa
PR:EMAIL                         	yb47620@um.edu.mo
PR:PHONE                         	88222534
PR:FUNDING_SOURCE                	University of Macau
#STUDY
ST:STUDY_TITLE                   	Investigate the False Discovery in Biomarker Research Using LC-HRMS based
ST:STUDY_TITLE                   	Untargeted Metabolomics Profiling
ST:STUDY_TYPE                    	mimicking metabolomics study
ST:STUDY_SUMMARY                 	Pooled human plasma was spiked separately with two different sets of 11
ST:STUDY_SUMMARY                 	metabolite standards (22 “true biomarkers”) to mimic plasma samples
ST:STUDY_SUMMARY                 	collected from diseased subjects and non-diseased subjects. Metabolite extracts
ST:STUDY_SUMMARY                 	of individual samples were subjected to biomarker discovery using LC-HRMS.
ST:INSTITUTE                     	University of Macau, Macau, China
ST:DEPARTMENT                    	Institute of Translational Medicine, Faculty of Health Sciences,
ST:LABORATORY                    	Pilot Laboratory
ST:LAST_NAME                     	ZHANG
ST:FIRST_NAME                    	pw
ST:ADDRESS                       	Taipa
ST:EMAIL                         	yb47620@um.edu.mo
ST:PHONE                         	88222534
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENDER                        	Pooled
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	A1	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A2	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A3	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A4	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A5	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A6	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A7	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A8	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A9	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A10	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A11	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	A12	SampleType:spiked SetA	
SUBJECT_SAMPLE_FACTORS           	-	B1	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B2	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B3	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B4	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B5	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B6	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B7	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B8	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B9	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B10	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B11	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	B12	SampleType:spiked SetB	
SUBJECT_SAMPLE_FACTORS           	-	Negative control1	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control2	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control3	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control4	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control5	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control6	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control7	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control8	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control9	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control10	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control11	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control12	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control13	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control14	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control15	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control16	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control17	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control18	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control19	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control20	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control21	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control22	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control23	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	Negative control24	SampleType:no treatment	
SUBJECT_SAMPLE_FACTORS           	-	QC1	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC2	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC3	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC4	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC5	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC6	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC7	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC8	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC9	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC10	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	QC11	SampleType:QC	
SUBJECT_SAMPLE_FACTORS           	-	Blank1	SampleType:blank	
SUBJECT_SAMPLE_FACTORS           	-	Blank2	SampleType:blank	
#COLLECTION
CO:COLLECTION_SUMMARY            	Pooled human potassium EDTA plasma was purchased from a commercial company
CO:COLLECTION_SUMMARY            	(Equitech-Bio Inc., Kerrville, TX, USA)
CO:SAMPLE_TYPE                   	Blood (plasma)
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Mimicking diseased plasma samples and non-diseased samples (12 samples for each
TR:TREATMENT_SUMMARY             	group) were spiked with Set A and Set B metabolite standards, respectively. Two
TR:TREATMENT_SUMMARY             	groups of negative control samples (12 samples for each group) were also
TR:TREATMENT_SUMMARY             	prepared, but replacing Set A and Set B metabolite standard mixtures with LC-MS
TR:TREATMENT_SUMMARY             	grade water. Information of the two sets of metabolite standards spiked into the
TR:TREATMENT_SUMMARY             	human pooled plasma. Set A (metabolite standard spiked concentration μM):
TR:TREATMENT_SUMMARY             	L-Leucine 250,Creatine 110, L-Histidine 160, L-Phenylalanine 130, D-(+)-Glucose
TR:TREATMENT_SUMMARY             	10000, L-Aspartic Acid 40, L-Valine 460, L-Alanine 660, L-Serine 280,
TR:TREATMENT_SUMMARY             	L-Carnitine 90, L-Glutamine 1180. Set B (metabolite standard spiked
TR:TREATMENT_SUMMARY             	concentration μM): Creatinine 150, L-Proline 480, Betaine 150, L-Glycine 460,
TR:TREATMENT_SUMMARY             	L-Arginine 220, L-Threonine 280, L-Tryptophan 100, L-Lysine 360, L-Glutamic Acid
TR:TREATMENT_SUMMARY             	170, L-Asparagine 80, Hypoxanthine 20.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	1. Preparation of Metabolite Standard Spiked Plasma Samples for the Biomarker
SP:SAMPLEPREP_SUMMARY            	Discovery Experiment Individual metabolite standard solutions were prepared in
SP:SAMPLEPREP_SUMMARY            	LC-MS grade water. Each set of metabolite standards was prepared separately by
SP:SAMPLEPREP_SUMMARY            	mixing equal volumes of 11 metabolite standards. The two metabolite mixtures was
SP:SAMPLEPREP_SUMMARY            	dried by speed vacuum at room temperature, and redissolved separately in the
SP:SAMPLEPREP_SUMMARY            	human pooled plasma to form two master spiked plasma samples. Each of the two
SP:SAMPLEPREP_SUMMARY            	master spiked plasma samples was divided into 12 identical aliquots (each 30
SP:SAMPLEPREP_SUMMARY            	µL), which were treated as plasma samples collected from 12 different people.
SP:SAMPLEPREP_SUMMARY            	The quality control (QC) sample was prepared by mixing equal amount of solutions
SP:SAMPLEPREP_SUMMARY            	of each spiked plasma sample, and it was used to check the stability of the
SP:SAMPLEPREP_SUMMARY            	system throughout the sequence analysis. 2. Preparation of Plasma Samples for
SP:SAMPLEPREP_SUMMARY            	the Negative Control Experiment Two groups of negative control samples (12
SP:SAMPLEPREP_SUMMARY            	identical samples for each group) were prepared according to the above strategy,
SP:SAMPLEPREP_SUMMARY            	but replacing Set A and Set B metabolite standard mixtures with LC-MS grade
SP:SAMPLEPREP_SUMMARY            	water. 3. Preparation of Blank Sample The solvent extraction blank samples were
SP:SAMPLEPREP_SUMMARY            	prepared according to the plasma metabolite extraction procedure, but replacing
SP:SAMPLEPREP_SUMMARY            	plasma with equal volume of MS grade water. 4. Plasma Metabolite Extraction
SP:SAMPLEPREP_SUMMARY            	Frozen pooled plasma samples were thawed at 4 oC and vortexed for 30 seconds
SP:SAMPLEPREP_SUMMARY            	before metabolite extraction. Thirty microliters of plasma were mixed with 120
SP:SAMPLEPREP_SUMMARY            	µL chilled methanol, and vortexed for 30 seconds. The mixture was incubated at
SP:SAMPLEPREP_SUMMARY            	4 oC for 20 minutes to precipitate the proteins. After incubation, the mixture
SP:SAMPLEPREP_SUMMARY            	was centrifuged at 14,000 g for 10 min to pellet the proteins. The supernatant
SP:SAMPLEPREP_SUMMARY            	were recovered as the metabolite extract, and stored at -80 oC before LC-HRMS
SP:SAMPLEPREP_SUMMARY            	analysis.
SP:PROCESSING_STORAGE_CONDITIONS 	4℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Individual metabolite extracts were separated on an amide HILIC (Accucore™ 150
CH:CHROMATOGRAPHY_SUMMARY        	Amide HILIC 2.1×150 mm, Thermo Fisher) column at a flow rate of 0.3 mL/min
CH:CHROMATOGRAPHY_SUMMARY        	using a UHPLC system (Dionex UltiMate 3000, Thermo Fisher).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                   	Unspecified
CH:FLOW_RATE                     	0.3 ml/min
CH:COLUMN_TEMPERATURE            	30
CH:METHODS_FILENAME              	Chromatography
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:INJECTION_TEMPERATURE         	8
CH:SAMPLE_INJECTION              	2 micro litter
CH:ANALYTICAL_TIME               	21 min
CH:CAPILLARY_VOLTAGE             	320
CH:PRECONDITIONING               	1h
#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                   	spray voltage, 3.5 kV; capillary temperature, 320 °C; sheath gas flow rate, 45;
MS:MS_COMMENTS                   	auxiliary gas flow rate, 25; heater temperature 350 °C; AGC, 3×106; maximum
MS:MS_COMMENTS                   	injection time, 200 ms; mass scan range, 50–750; full MS resolution, 70,000
MS:MS_COMMENTS                   	FWHM at m/z 200; spectrum data type, profile.
MS:MS_RESULTS_FILE               	ST001132_AN001857_Results.txt	UNITS:ARBITRARY	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END