#METABOLOMICS WORKBENCH epannkuk_20220414_115412 DATATRACK_ID:3202 STUDY_ID:ST002175 ANALYSIS_ID:AN003563
VERSION                          	1
CREATED_ON                       	10-26-2022
#PROJECT
PR:PROJECT_TITLE                 	Effect of external high-dose rate radiation on mouse biofluid metabolomics
PR:PROJECT_SUMMARY               	In the event of an improvised nuclear device (IND), a complex IR exposure will
PR:PROJECT_SUMMARY               	occur consisting of both low (LDR) and high-dose rates (HDR). We have previously
PR:PROJECT_SUMMARY               	addressed LDR exposures from internal emitters or externally deposited
PR:PROJECT_SUMMARY               	radionuclides on biofluid small molecule signatures, but further research on the
PR:PROJECT_SUMMARY               	HDR component is required. Here, we exposed 8 − 10 week old male C57BL/6 mice
PR:PROJECT_SUMMARY               	to a cumulative dose of 3 Gy using a reference dose rate of 0.7 Gy/min or a HDR
PR:PROJECT_SUMMARY               	of 7 Gy/sec, collected urine and serum at 1 and 7 d, then compared the
PR:PROJECT_SUMMARY               	metabolite signatures using a untargeted (urine) approache with liquid
PR:PROJECT_SUMMARY               	chromatography mass spectrometry platforms.
PR:INSTITUTE                     	Georgetown University
PR:LAST_NAME                     	Pannkuk
PR:FIRST_NAME                    	Evan
PR:ADDRESS                       	3970 Reservoir Rd, NW New Research Building E504
PR:EMAIL                         	elp44@georgetown.edu
PR:PHONE                         	2026875650
PR:PUBLICATIONS                  	https://www.mdpi.com/2218-1989/12/6/520
PR:DOI                           	http://dx.doi.org/10.21228/M8TH8J
#STUDY
ST:STUDY_TITLE                   	Effect of external high-dose rate radiation on mouse biofluid metabolomic
ST:STUDY_TITLE                   	signatures
ST:STUDY_SUMMARY                 	In the event of an improvised nuclear device (IND), a complex IR exposure will
ST:STUDY_SUMMARY                 	occur consisting of both low (LDR) and high-dose rates (HDR). We have previously
ST:STUDY_SUMMARY                 	addressed LDR exposures from internal emitters or externally deposited
ST:STUDY_SUMMARY                 	radionuclides on biofluid small molecule signatures, but further research on the
ST:STUDY_SUMMARY                 	HDR component is required. Here, we exposed 8 − 10 week old male C57BL/6 mice
ST:STUDY_SUMMARY                 	to a cumulative dose of 3 Gy using a reference dose rate of 0.7 Gy/min or a HDR
ST:STUDY_SUMMARY                 	of 7 Gy/sec, collected urine and serum at 1 and 7 d, then compared the
ST:STUDY_SUMMARY                 	metabolite signatures using either untargeted (urine) or targeted (serum)
ST:STUDY_SUMMARY                 	approaches with liquid chromatography mass spectrometry platforms.
ST:INSTITUTE                     	Georgetown University
ST:LAST_NAME                     	Pannkuk
ST:FIRST_NAME                    	Evan
ST:ADDRESS                       	3970 Reservoir Rd, NW New Research Building E504
ST:EMAIL                         	elp44@georgetown.edu
ST:PHONE                         	2026875650
ST:SUBMIT_DATE                   	2022-04-14
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:AGE_OR_AGE_RANGE              	8 - 10 weeks
SU:GENDER                        	Male
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	18-2	113	Factor:CTL | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_146
SUBJECT_SAMPLE_FACTORS           	16-2	13	Factor:CTL | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_023
SUBJECT_SAMPLE_FACTORS           	13-2	14	Factor:CTL | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_024
SUBJECT_SAMPLE_FACTORS           	15-2	16	Factor:CTL | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_026
SUBJECT_SAMPLE_FACTORS           	14-2	62	Factor:CTL | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_082
SUBJECT_SAMPLE_FACTORS           	17-2	84	Factor:CTL | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_112
SUBJECT_SAMPLE_FACTORS           	15-2	115	Factor:CTL | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_151
SUBJECT_SAMPLE_FACTORS           	13-2	127	Factor:CTL | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_166
SUBJECT_SAMPLE_FACTORS           	18-2	132	Factor:CTL | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_170
SUBJECT_SAMPLE_FACTORS           	14-2	5	Factor:CTL | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_012
SUBJECT_SAMPLE_FACTORS           	16-2	70	Factor:CTL | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_093
SUBJECT_SAMPLE_FACTORS           	17-2	85	Factor:CTL | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_113
SUBJECT_SAMPLE_FACTORS           	33-2	120	Factor:CTL | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_156
SUBJECT_SAMPLE_FACTORS           	32-2	24	Factor:CTL | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_037
SUBJECT_SAMPLE_FACTORS           	34-2	51	Factor:CTL | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_069
SUBJECT_SAMPLE_FACTORS           	36-2	78	Factor:CTL | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_104
SUBJECT_SAMPLE_FACTORS           	33-2	116	Factor:CTL | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_152
SUBJECT_SAMPLE_FACTORS           	32-2	133	Factor:CTL | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_171
SUBJECT_SAMPLE_FACTORS           	36-2	142	Factor:CTL | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_182
SUBJECT_SAMPLE_FACTORS           	31-2	146	Factor:CTL | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_186
SUBJECT_SAMPLE_FACTORS           	34-2	18	Factor:CTL | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_028
SUBJECT_SAMPLE_FACTORS           	35-2	73	Factor:CTL | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_099
SUBJECT_SAMPLE_FACTORS           	1-2	108	Factor:HDR | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_142
SUBJECT_SAMPLE_FACTORS           	6-2	111	Factor:HDR | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_144
SUBJECT_SAMPLE_FACTORS           	5-2	114	Factor:HDR | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_147
SUBJECT_SAMPLE_FACTORS           	4-2	117	Factor:HDR | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_153
SUBJECT_SAMPLE_FACTORS           	3-2	139	Factor:HDR | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_179
SUBJECT_SAMPLE_FACTORS           	2-2	48	Factor:HDR | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_066
SUBJECT_SAMPLE_FACTORS           	1-2	122	Factor:HDR | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_158
SUBJECT_SAMPLE_FACTORS           	4-2	128	Factor:HDR | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_167
SUBJECT_SAMPLE_FACTORS           	3-2	45	Factor:HDR | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_064
SUBJECT_SAMPLE_FACTORS           	2-2	57	Factor:HDR | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_077
SUBJECT_SAMPLE_FACTORS           	6-2	86	Factor:HDR | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_114
SUBJECT_SAMPLE_FACTORS           	5-2	88	Factor:HDR | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_116
SUBJECT_SAMPLE_FACTORS           	19-2	103	Factor:HDR | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_134
SUBJECT_SAMPLE_FACTORS           	22-2	11	Factor:HDR | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_018
SUBJECT_SAMPLE_FACTORS           	21-2	129	Factor:HDR | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_168
SUBJECT_SAMPLE_FACTORS           	24-2	145	Factor:HDR | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_185
SUBJECT_SAMPLE_FACTORS           	23-2	54	Factor:HDR | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_075
SUBJECT_SAMPLE_FACTORS           	20-2	92	Factor:HDR | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_120
SUBJECT_SAMPLE_FACTORS           	24-2	137	Factor:HDR | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_177
SUBJECT_SAMPLE_FACTORS           	20-2	141	Factor:HDR | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_181
SUBJECT_SAMPLE_FACTORS           	22-2	41	Factor:HDR | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_057
SUBJECT_SAMPLE_FACTORS           	21-2	60	Factor:HDR | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_080
SUBJECT_SAMPLE_FACTORS           	23-2	64	Factor:HDR | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_087
SUBJECT_SAMPLE_FACTORS           	9-2	106	Factor:REF | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_140
SUBJECT_SAMPLE_FACTORS           	8-2	33	Factor:REF | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_049
SUBJECT_SAMPLE_FACTORS           	11-2	38	Factor:REF | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_054
SUBJECT_SAMPLE_FACTORS           	10-2	65	Factor:REF | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_088
SUBJECT_SAMPLE_FACTORS           	12-2	68	Factor:REF | Factor:D1 | Factor:post	RAW_FILE_NAME=POS_091
SUBJECT_SAMPLE_FACTORS           	9-2	121	Factor:REF | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_157
SUBJECT_SAMPLE_FACTORS           	10-2	151	Factor:REF | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_190
SUBJECT_SAMPLE_FACTORS           	12-2	22	Factor:REF | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_035
SUBJECT_SAMPLE_FACTORS           	8-2	34	Factor:REF | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_050
SUBJECT_SAMPLE_FACTORS           	11-2	61	Factor:REF | Factor:D1 | Factor:pre	RAW_FILE_NAME=POS_081
SUBJECT_SAMPLE_FACTORS           	25-2	131	Factor:REF | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_169
SUBJECT_SAMPLE_FACTORS           	30-2	150	Factor:REF | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_189
SUBJECT_SAMPLE_FACTORS           	27-2	26	Factor:REF | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_039
SUBJECT_SAMPLE_FACTORS           	28-2	30	Factor:REF | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_043
SUBJECT_SAMPLE_FACTORS           	26-2	40	Factor:REF | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_056
SUBJECT_SAMPLE_FACTORS           	29-2	96	Factor:REF | Factor:D7 | Factor:post	RAW_FILE_NAME=POS_127
SUBJECT_SAMPLE_FACTORS           	25-2	125	Factor:REF | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_164
SUBJECT_SAMPLE_FACTORS           	27-2	136	Factor:REF | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_173
SUBJECT_SAMPLE_FACTORS           	26-2	17	Factor:REF | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_027
SUBJECT_SAMPLE_FACTORS           	29-2	23	Factor:REF | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_036
SUBJECT_SAMPLE_FACTORS           	30-2	77	Factor:REF | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_103
SUBJECT_SAMPLE_FACTORS           	28-2	94	Factor:REF | Factor:D7 | Factor:pre	RAW_FILE_NAME=POS_125
#COLLECTION
CO:COLLECTION_SUMMARY            	Urine was collected after irradiation
CO:SAMPLE_TYPE                   	Urine
CO:COLLECTION_METHOD             	Spot Urine
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Male 8 – 10 week old C57BL/6 mice were obtained from Charles River
TR:TREATMENT_SUMMARY             	Laboratories (Frederick, MD, USA) and were irradiated using the FLASH irradiator
TR:TREATMENT_SUMMARY             	at the Radiological Research Accelerator Facility [16] (Figure S1). This novel
TR:TREATMENT_SUMMARY             	irradiator is based on a Clinac 2100C (Varian Medical Systems, Corona, CA, USA)
TR:TREATMENT_SUMMARY             	where the pulse delivery is controlled using in house software. All irradiations
TR:TREATMENT_SUMMARY             	were performed using 9 MeV electrons with no scatterer or flattening filter. For
TR:TREATMENT_SUMMARY             	these experiments, mice were placed in a 72 mm x 41mm x41mm acrylic box in which
TR:TREATMENT_SUMMARY             	air holes had been drilled (The Container Store, Coppell, TX, USA). For 0.7
TR:TREATMENT_SUMMARY             	Gy/min irradiations, mice (n=6) were individually placed at 120 cm above the
TR:TREATMENT_SUMMARY             	clinac head and irradiation delivered at 3.25 pulses per second. In this
TR:TREATMENT_SUMMARY             	configuration, 3 Gy was delivered in 580 pulses. For 7 Gy/sec mice (n=6) were
TR:TREATMENT_SUMMARY             	individually placed 20cm above the clinac head (Figure S1) and dose delivered at
TR:TREATMENT_SUMMARY             	180 pulses/sec after allowing 20 sec where the acceleration and electron source
TR:TREATMENT_SUMMARY             	were both on but operated asynchronously so that no beam is delivered. In this
TR:TREATMENT_SUMMARY             	configuration, 3 Gy was delivered in 78 pulses. Dosimetry was performed prior to
TR:TREATMENT_SUMMARY             	irradiation using a NIST-traceable Advanced Marcus Ion Chamber (AMIC) and Unidos
TR:TREATMENT_SUMMARY             	E electrometer (PTW, Freiburg, Germany). Verification of dosimetry was performed
TR:TREATMENT_SUMMARY             	using OBT3 radiochromic film (Ashland Specialty Chemicals, Wayne, NJ, USA).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	urine (20 μl) was deproteinized (80 μl 50% cold acetonitrile) with internal
SP:SAMPLEPREP_SUMMARY            	standards (2 μM debrisoquine [M+H]+ = 176.1188; 30 μM 4-nitrobenzoic acid
SP:SAMPLEPREP_SUMMARY            	[M-H]- = 166.0141), vortexed, incubated on ice (10 min), and centrifuged for 10
SP:SAMPLEPREP_SUMMARY            	min (max speed, 4 °C). A 1 μl aliquot of each sample was combined for a
SP:SAMPLEPREP_SUMMARY            	quality control (QC) sample.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Mobile phases consisted of the following: solvent A (water/0.1% formic acid
CH:CHROMATOGRAPHY_SUMMARY        	[FA]) and solvent B (acetonitrile/0.1% FA). The gradient for urine was (solvent
CH:CHROMATOGRAPHY_SUMMARY        	A and B) 4.0 min 5% B, 4.0 min 20% B, 5.1 min 95% B, and 1.9 min 5% B at a flow
CH:CHROMATOGRAPHY_SUMMARY        	rate of 0.5 ml/min, column temp 40 °C.
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters Acquity BEH C18 (50 x 2.1mm, 1.7 um)
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Synapt G2 S QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	Samples were injected (2 μl) into a Waters Acquity Ultra Performance Liquid
MS:MS_COMMENTS                   	Chromatography (UPLC) with a BEH C18 1.7 μm, 2.1 x 50 mm column and coupled to
MS:MS_COMMENTS                   	a Xevo® G2-S quadrupole time-of-flight (QTOF) MS (Waters, Milford, MA, USA).
MS:MS_COMMENTS                   	Positive and negative electrospray ionization (ESI) data-independent modes were
MS:MS_COMMENTS                   	used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- =
MS:MS_COMMENTS                   	554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage
MS:MS_COMMENTS                   	2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow
MS:MS_COMMENTS                   	1000 L/Hr.
MS:ION_MODE                      	POSITIVE
MS:MS_RESULTS_FILE               	ST002175_AN003563_Results.txt	UNITS:peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END