#METABOLOMICS WORKBENCH EmilyLette_20210225_014138 DATATRACK_ID:2509 STUDY_ID:ST001721 ANALYSIS_ID:AN002804 PROJECT_ID:PR001103
VERSION             	1
CREATED_ON             	March 15, 2021, 12:03 pm
#PROJECT
PR:PROJECT_TITLE                 	Detecting sex-related changes to the metabolome of a critically endangered
PR:PROJECT_TITLE                 	freshwater crayfish during the mating season
PR:PROJECT_TYPE                  	MS analysis of crustacean haemolymph
PR:PROJECT_SUMMARY               	Captive breeding is a vital tool in the conservation of highly endangered
PR:PROJECT_SUMMARY               	species, as it is for the Margaret River hairy marron, Cherax tenuimanus, from
PR:PROJECT_SUMMARY               	the south west of Australia. A close relative, Cherax cainii, has almost
PR:PROJECT_SUMMARY               	completely displaced C. tenuimanus in the wild and is a successful aquaculture
PR:PROJECT_SUMMARY               	species, whereas C. tenuimanus has performed poorly in captivity. We used
PR:PROJECT_SUMMARY               	untargeted liquid chromatography-mass spectrometry to obtain metabolomic
PR:PROJECT_SUMMARY               	profiles of female and male C. tenuimanus held in controlled aquarium conditions
PR:PROJECT_SUMMARY               	during their reproductive period. Using repeated haemolymph sampling we tracked
PR:PROJECT_SUMMARY               	the metabolomic profiles of animals just prior to and for a period of up to 34
PR:PROJECT_SUMMARY               	days after pairing with a similar sized potential mate. We identified 54
PR:PROJECT_SUMMARY               	reproducible annotated metabolites including amino acids, fatty acids, biogenic
PR:PROJECT_SUMMARY               	amines, purine and pyrimidine metabolites and excretion metabolites.
PR:PROJECT_SUMMARY               	Hierarchical clustering analysis distinguished five metabolite clusters.
PR:PROJECT_SUMMARY               	Principal component-canonical variate analysis clearly distinguished females
PR:PROJECT_SUMMARY               	from males, both unpaired and paired; similar trends in profile changes in both
PR:PROJECT_SUMMARY               	sexes after pairing; and a striking shift in males upon pairing. We discuss
PR:PROJECT_SUMMARY               	three main patterns of metabolomic responses: differentiation between sexes;
PR:PROJECT_SUMMARY               	reactive responses to the disturbance of pairing; and convergent response to the
PR:PROJECT_SUMMARY               	disturbance of pairing for males. Females generally had higher concentrations of
PR:PROJECT_SUMMARY               	metabolites involved in metabolic rate, mobilisation of energy stores and
PR:PROJECT_SUMMARY               	stress. Responses to the disturbance of pairing were also related to elevated
PR:PROJECT_SUMMARY               	stress. Females were mobilising lipid stores to deposit yolk, whereas males had
PR:PROJECT_SUMMARY               	a rapid and strong response to pairing, with shifts in metabolites associated
PR:PROJECT_SUMMARY               	with gonad development and communication, indicating males could complete
PR:PROJECT_SUMMARY               	reproductive readiness only once paired with a female. The metabolomic profiles
PR:PROJECT_SUMMARY               	support a previously proposed potential mechanism for displacement of C.
PR:PROJECT_SUMMARY               	tenuimanus by C. cainii in the wild and identify several biomarkers for testing
PR:PROJECT_SUMMARY               	hypotheses regarding reproductive success using targeted metabolomics.
PR:INSTITUTE                     	Edith Cowan University
PR:DEPARTMENT                    	School of Science
PR:LAST_NAME                     	Lette
PR:FIRST_NAME                    	Emily
PR:ADDRESS                       	270 Joondalup Drive, Joondalup, WA, 6027, Australia
PR:EMAIL                         	e.lette@ecu.edu.au
PR:PHONE                         	+61 8 6304 5513
#STUDY
ST:STUDY_TITLE                   	Detecting sex-related changes to the metabolome of a critically endangered
ST:STUDY_TITLE                   	freshwater crayfish during the mating season
ST:STUDY_TYPE                    	LC-MS analysis of crustacean haemolymph
ST:STUDY_SUMMARY                 	Captive breeding is a vital tool in the conservation of highly endangered
ST:STUDY_SUMMARY                 	species, as it is for the Margaret River hairy marron, Cherax tenuimanus, from
ST:STUDY_SUMMARY                 	the south west of Australia. A close relative, Cherax cainii, has almost
ST:STUDY_SUMMARY                 	completely displaced C. tenuimanus in the wild and is a successful aquaculture
ST:STUDY_SUMMARY                 	species, whereas C. tenuimanus has performed poorly in captivity. We used
ST:STUDY_SUMMARY                 	untargeted liquid chromatography-mass spectrometry to obtain metabolomic
ST:STUDY_SUMMARY                 	profiles of female and male C. tenuimanus held in controlled aquarium conditions
ST:STUDY_SUMMARY                 	during their reproductive period. Using repeated haemolymph sampling we tracked
ST:STUDY_SUMMARY                 	the metabolomic profiles of animals just prior to and for a period of up to 34
ST:STUDY_SUMMARY                 	days after pairing with a similar sized potential mate. We identified 54
ST:STUDY_SUMMARY                 	reproducible annotated metabolites including amino acids, fatty acids, biogenic
ST:STUDY_SUMMARY                 	amines, purine and pyrimidine metabolites and excretion metabolites.
ST:STUDY_SUMMARY                 	Hierarchical clustering analysis distinguished five metabolite clusters.
ST:STUDY_SUMMARY                 	Principal component-canonical variate analysis clearly distinguished females
ST:STUDY_SUMMARY                 	from males, both unpaired and paired; similar trends in profile changes in both
ST:STUDY_SUMMARY                 	sexes after pairing; and a striking shift in males upon pairing. We discuss
ST:STUDY_SUMMARY                 	three main patterns of metabolomic responses: differentiation between sexes;
ST:STUDY_SUMMARY                 	reactive responses to the disturbance of pairing; and convergent response to the
ST:STUDY_SUMMARY                 	disturbance of pairing for males. Females generally had higher concentrations of
ST:STUDY_SUMMARY                 	metabolites involved in metabolic rate, mobilisation of energy stores and
ST:STUDY_SUMMARY                 	stress. Responses to the disturbance of pairing were also related to elevated
ST:STUDY_SUMMARY                 	stress. Females were mobilising lipid stores to deposit yolk, whereas males had
ST:STUDY_SUMMARY                 	a rapid and strong response to pairing, with shifts in metabolites associated
ST:STUDY_SUMMARY                 	with gonad development and communication, indicating males could complete
ST:STUDY_SUMMARY                 	reproductive readiness only once paired with a female. The metabolomic profiles
ST:STUDY_SUMMARY                 	support a previously proposed potential mechanism for displacement of C.
ST:STUDY_SUMMARY                 	tenuimanus by C. cainii in the wild and identify several biomarkers for testing
ST:STUDY_SUMMARY                 	hypotheses regarding reproductive success using targeted metabolomics.
ST:INSTITUTE                     	Edith Cowan University
ST:DEPARTMENT                    	School of Science
ST:LAST_NAME                     	Lette
ST:FIRST_NAME                    	Emily
ST:ADDRESS                       	270 Joondalup Drive, Joondalup, WA, 6027, Australia
ST:EMAIL                         	e.lette@ecu.edu.au
ST:PHONE                         	+61 8 6304 5513
ST:TOTAL_SUBJECTS                	10
ST:NUM_MALES                     	5
ST:NUM_FEMALES                   	5
#SUBJECT
SU:SUBJECT_TYPE                  	Invertebrate
SU:SUBJECT_SPECIES               	Cherax tenuimanus
SU:TAXONOMY_ID                   	99755
SU:WEIGHT_OR_WEIGHT_RANGE        	116g-198g
SU:GENDER                        	Male and female
#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           	-	QC9	Sex:- | Timepoint:-	RAW_FILE_NAME=Area_QE_2018_Q2_0872_QC_raw_F1_;Area_QE_2018_Q2_0971_QC_raw_F1_
SUBJECT_SAMPLE_FACTORS           	-	QC10	Sex:- | Timepoint:-	RAW_FILE_NAME=Area_QE_2018_Q2_0873_QC_raw_F2_;Area_QE_2018_Q2_0972_QC_raw_F2_
SUBJECT_SAMPLE_FACTORS           	-	FH1_4	Sex:Female | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0874_Sample_raw_F3_;Area_QE_2018_Q2_0973_Sample_raw_F3_
SUBJECT_SAMPLE_FACTORS           	-	FH1_2	Sex:Female | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0875_Sample_raw_F4_;Area_QE_2018_Q2_0974_Sample_raw_F4_
SUBJECT_SAMPLE_FACTORS           	-	FH1_3	Sex:Female | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0876_Sample_raw_F5_;Area_QE_2018_Q2_0975_Sample_raw_F5_
SUBJECT_SAMPLE_FACTORS           	-	FH1_1	Sex:Female | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0877_Sample_raw_F6_;Area_QE_2018_Q2_0976_Sample_raw_F6_
SUBJECT_SAMPLE_FACTORS           	-	FH2_1	Sex:Female | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0878_Sample_raw_F7_;Area_QE_2018_Q2_0977_Sample_raw_F7_
SUBJECT_SAMPLE_FACTORS           	-	FH2_3	Sex:Female | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0879_Sample_raw_F8_;Area_QE_2018_Q2_0978_Sample_raw_F8_
SUBJECT_SAMPLE_FACTORS           	-	FH2_2	Sex:Female | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0880_Sample_raw_F9_;Area_QE_2018_Q2_0979_Sample_raw_F9_
SUBJECT_SAMPLE_FACTORS           	-	FH2_4	Sex:Female | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0881_Sample_raw_F10_;Area_QE_2018_Q2_0980_Sample_raw_F10_
SUBJECT_SAMPLE_FACTORS           	-	QC11	Sex:- | Timepoint:-	RAW_FILE_NAME=Area_QE_2018_Q2_0882_QC_raw_F11_;Area_QE_2018_Q2_0981_QC_raw_F11_
SUBJECT_SAMPLE_FACTORS           	-	FH3_3	Sex:Female | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0883_Sample_raw_F12_;Area_QE_2018_Q2_0982_Sample_raw_F12_
SUBJECT_SAMPLE_FACTORS           	-	FH3_1	Sex:Female | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0884_Sample_raw_F13_;Area_QE_2018_Q2_0983_Sample_raw_F13_
SUBJECT_SAMPLE_FACTORS           	-	FH3_2	Sex:Female | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0885_Sample_raw_F14_;Area_QE_2018_Q2_0984_Sample_raw_F14_
SUBJECT_SAMPLE_FACTORS           	-	FH3_4	Sex:Female | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0886_Sample_raw_F15_;Area_QE_2018_Q2_0985_Sample_raw_F15_
SUBJECT_SAMPLE_FACTORS           	-	FH4_2	Sex:Female | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0887_Sample_raw_F16_;Area_QE_2018_Q2_0986_Sample_raw_F16_
SUBJECT_SAMPLE_FACTORS           	-	FH4_4	Sex:Female | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0888_Sample_raw_F17_;Area_QE_2018_Q2_0987_Sample_raw_F17_
SUBJECT_SAMPLE_FACTORS           	-	FH4_1	Sex:Female | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0889_Sample_raw_F18_;Area_QE_2018_Q2_0988_Sample_raw_F18_
SUBJECT_SAMPLE_FACTORS           	-	FH4_3	Sex:Female | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0890_Sample_raw_F19_;Area_QE_2018_Q2_0989_Sample_raw_F19_
SUBJECT_SAMPLE_FACTORS           	-	QC12	Sex:- | Timepoint:-	RAW_FILE_NAME=Area_QE_2018_Q2_0891_QC_raw_F20_;Area_QE_2018_Q2_0990_QC_raw_F20_
SUBJECT_SAMPLE_FACTORS           	-	FH5_3	Sex:Female | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0892_Sample_raw_F21_;Area_QE_2018_Q2_0991_Sample_raw_F21_
SUBJECT_SAMPLE_FACTORS           	-	FH5_2	Sex:Female | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0893_Sample_raw_F22_;Area_QE_2018_Q2_0992_Sample_raw_F22_
SUBJECT_SAMPLE_FACTORS           	-	FH5_1	Sex:Female | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0894_Sample_raw_F23_;Area_QE_2018_Q2_0993_Sample_raw_F23_
SUBJECT_SAMPLE_FACTORS           	-	FH5_4	Sex:Female | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0895_Sample_raw_F24_;Area_QE_2018_Q2_0994_Sample_raw_F24_
SUBJECT_SAMPLE_FACTORS           	-	MH1_4	Sex:Male | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0896_Sample_raw_F25_;Area_QE_2018_Q2_0995_Sample_raw_F25_
SUBJECT_SAMPLE_FACTORS           	-	MH1_3	Sex:Male | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0897_Sample_raw_F26_;Area_QE_2018_Q2_0996_Sample_raw_F26_
SUBJECT_SAMPLE_FACTORS           	-	MH1_2	Sex:Male | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0898_Sample_raw_F27_;Area_QE_2018_Q2_0997_Sample_raw_F27_
SUBJECT_SAMPLE_FACTORS           	-	MH1_1	Sex:Male | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0899_Sample_raw_F28_;Area_QE_2018_Q2_0998_Sample_raw_F28_
SUBJECT_SAMPLE_FACTORS           	-	QC13	Sex:- | Timepoint:-	RAW_FILE_NAME=Area_QE_2018_Q2_0900_QC_raw_F29_;Area_QE_2018_Q2_0999_QC_raw_F29_
SUBJECT_SAMPLE_FACTORS           	-	MH2_4	Sex:Male | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0901_Sample_raw_F30_;Area_QE_2018_Q2_1000_Sample_raw_F30_
SUBJECT_SAMPLE_FACTORS           	-	MH2_3	Sex:Male | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0902_Sample_raw_F31_;Area_QE_2018_Q2_1001_Sample_raw_F31_
SUBJECT_SAMPLE_FACTORS           	-	MH2_1	Sex:Male | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0903_Sample_raw_F32_;Area_QE_2018_Q2_1002_Sample_raw_F32_
SUBJECT_SAMPLE_FACTORS           	-	MH2_2	Sex:Male | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0904_Sample_raw_F33_;Area_QE_2018_Q2_1003_Sample_raw_F33_
SUBJECT_SAMPLE_FACTORS           	-	MH3_4	Sex:Male | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0905_Sample_raw_F34_;Area_QE_2018_Q2_1004_Sample_raw_F34_
SUBJECT_SAMPLE_FACTORS           	-	MH3_3	Sex:Male | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0906_Sample_raw_F35_;Area_QE_2018_Q2_1005_Sample_raw_F35_
SUBJECT_SAMPLE_FACTORS           	-	MH3_2	Sex:Male | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0907_Sample_raw_F36_;Area_QE_2018_Q2_1006_Sample_raw_F36_
SUBJECT_SAMPLE_FACTORS           	-	MH3_1	Sex:Male | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0908_Sample_raw_F37_;Area_QE_2018_Q2_1007_Sample_raw_F37_
SUBJECT_SAMPLE_FACTORS           	-	QC14	Sex:- | Timepoint:-	RAW_FILE_NAME=Area_QE_2018_Q2_0909_QC_raw_F38_;Area_QE_2018_Q2_1008_QC_raw_F38_
SUBJECT_SAMPLE_FACTORS           	-	MH4_2	Sex:Male | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0910_Sample_raw_F39_;Area_QE_2018_Q2_1009_Sample_raw_F39_
SUBJECT_SAMPLE_FACTORS           	-	MH4_4	Sex:Male | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0911_Sample_raw_F40_;Area_QE_2018_Q2_1010_Sample_raw_F40_
SUBJECT_SAMPLE_FACTORS           	-	MH4_1	Sex:Male | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0912_Sample_raw_F41_;Area_QE_2018_Q2_1011_Sample_raw_F41_
SUBJECT_SAMPLE_FACTORS           	-	MH4_3	Sex:Male | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0913_Sample_raw_F42_;Area_QE_2018_Q2_1012_Sample_raw_F42_
SUBJECT_SAMPLE_FACTORS           	-	MH5_3	Sex:Male | Timepoint:3	RAW_FILE_NAME=Area_QE_2018_Q2_0914_Sample_raw_F43_;Area_QE_2018_Q2_1013_Sample_raw_F43_
SUBJECT_SAMPLE_FACTORS           	-	MH5_4	Sex:Male | Timepoint:4	RAW_FILE_NAME=Area_QE_2018_Q2_0915_Sample_raw_F44_;Area_QE_2018_Q2_1014_Sample_raw_F44_
SUBJECT_SAMPLE_FACTORS           	-	MH5_1	Sex:Male | Timepoint:1	RAW_FILE_NAME=Area_QE_2018_Q2_0916_Sample_raw_F45_;Area_QE_2018_Q2_1015_Sample_raw_F45_
SUBJECT_SAMPLE_FACTORS           	-	MH5_2	Sex:Male | Timepoint:2	RAW_FILE_NAME=Area_QE_2018_Q2_0917_Sample_raw_F46_;Area_QE_2018_Q2_1016_Sample_raw_F46_
SUBJECT_SAMPLE_FACTORS           	-	QC15	Sex:- | Timepoint:-	RAW_FILE_NAME=Area_QE_2018_Q2_0918_QC_raw_F47_;Area_QE_2018_Q2_1017_QC_raw_F47_
#COLLECTION
CO:COLLECTION_SUMMARY            	All haemolymph collections for all animals occurred at the same time of day and
CO:COLLECTION_SUMMARY            	the animals were handled in the same order on each of the four collection dates.
CO:COLLECTION_SUMMARY            	Haemolymph (200mL) was extracted from the ventral sinus of each crayfish using a
CO:COLLECTION_SUMMARY            	21G needle and 1mL syringe inserted into the soft tissue at the base of the 5th
CO:COLLECTION_SUMMARY            	pereopod. Haemolymph was added to 2mL Eppendorf tubes preloaded with 600µL
CO:COLLECTION_SUMMARY            	LC-MS grade acetonitrile (Optima, Thermo Fisher Scientific, AUS) containing
CO:COLLECTION_SUMMARY            	deuterated internal standards (d8- valine, d9 –trimethylamine-N-oxide (TMAO) ,
CO:COLLECTION_SUMMARY            	d3-leucine, d6-trans-cinnamic acid, d5-tryptophan, 1g/mL) from Cambridge
CO:COLLECTION_SUMMARY            	Isotope Laboratories (Cambridge, MA, USA) and stored on ice. Samples were mixed
CO:COLLECTION_SUMMARY            	at 1400rpm (Thermal Mixer, Thermo Fisher Scientific, AUS) for 60 seconds at
CO:COLLECTION_SUMMARY            	4°C, then centrifuged (Heraeus Megafuge 8R, Thermo Fisher Scientific, AUS) for
CO:COLLECTION_SUMMARY            	20 minutes (1800  g) at 4°C. After centrifuging, 100µL of supernatant was
CO:COLLECTION_SUMMARY            	aliquoted into five separate vials. The samples were then dried using a
CO:COLLECTION_SUMMARY            	SpeedVac centrifugal vacuum concentrator (Thermo Fisher Scientific, AUS). The
CO:COLLECTION_SUMMARY            	dried samples were stored at -80°C for subsequent metabolomics analysis.
CO:COLLECTION_PROTOCOL_FILENAME  	EmilyLette_20210225_014138_PR_CO_Methods__Lette_marron.pdf
CO:SAMPLE_TYPE                   	Hemolymph
CO:COLLECTION_METHOD             	aspiration
CO:COLLECTION_LOCATION           	ventral sinus accessed from soft tissue at base of 5th pereopod
CO:STORAGE_CONDITIONS            	-80℃
CO:COLLECTION_VIALS              	2mL Eppendorf tubes
CO:STORAGE_VIALS                 	2mL Eppendorf tubes
#TREATMENT
TR:TREATMENT_SUMMARY             	Cherax tenuimanus (n=10) were housed in aquaria as individuals prior to sampling
TR:TREATMENT_SUMMARY             	at timepoint 1. After timepoint 1 they were placed in pairs with a potential
TR:TREATMENT_SUMMARY             	mate of the opposite sex and housed in aquaria over the next four weeks which
TR:TREATMENT_SUMMARY             	included timepoints 2-4.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The dried haemolymph samples were reconstituted using 100µL of LC-MS water
SP:SAMPLEPREP_SUMMARY            	containing 0.1% Formic acid. Samples were manually swirled, then placed in a
SP:SAMPLEPREP_SUMMARY            	thermomixer for 2 minutes at 4oC, before being centrifuged at (1800  g) for 5
SP:SAMPLEPREP_SUMMARY            	minutes at 4oC. Next, 40µL of the supernatant was transferred into LC-MS amber
SP:SAMPLEPREP_SUMMARY            	vials with inserts and placed in the autosampler kept at 6oC. The order in which
SP:SAMPLEPREP_SUMMARY            	samples were analysed was randomised to avoid any potential instrument bias. A
SP:SAMPLEPREP_SUMMARY            	pooled quality control (QC) sample was prepared by adding 40 µL from each
SP:SAMPLEPREP_SUMMARY            	reconstituted sample to a single Eppendorf tube, which was then mixed to
SP:SAMPLEPREP_SUMMARY            	homogenise in a thermal mixer and centrifuged as above. This pooled sample was
SP:SAMPLEPREP_SUMMARY            	aliquoted (40µL) into LC-MS amber vials to create 16 QC samples and placed into
SP:SAMPLEPREP_SUMMARY            	the autosampler tray kept at 6°C ready for analysis. Samples were analysed
SP:SAMPLEPREP_SUMMARY            	within 24 hours from preparation. At the start of the analytical batch, a
SP:SAMPLEPREP_SUMMARY            	solvent blank, matrix blank, and ten conditioning QC samples were analysed
SP:SAMPLEPREP_SUMMARY            	(Broadhurst et al., 2018). QC samples were then injected after every fifth
SP:SAMPLEPREP_SUMMARY            	marron haemolymph sample with two QCs analysed at the end of the batch,
SP:SAMPLEPREP_SUMMARY            	following the standard protocols for metabolomics (Broadhurst et al., 2018).
SP:PROCESSING_STORAGE_CONDITIONS 	Described in summary
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000 RS
CH:COLUMN_NAME                   	Thermo Hypersil GOLD C18 (100 x 2.1mm,1.9µm)
CH:FLOW_GRADIENT                 	isocratic at 99% A: 1 min; 50% B:1-2 min; linear increase to 99% B: over 7min;
CH:FLOW_GRADIENT                 	99% B: 2min; initial conditions returned over 2 min; 100% A: 3 min.
CH:FLOW_RATE                     	0.3mL/min
CH:COLUMN_TEMPERATURE            	45
CH:METHODS_FILENAME              	Methods_Lette_marron
CH:SOLVENT_A                     	0.1% formic acid in LC-MS water
CH:SOLVENT_B                     	0.1% formic acid in LC-MS acetonitrile
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:ANALYSIS_PROTOCOL_FILE        	Methods_Lette_marron
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Full scans with data-dependent tandem mass spectrometry were acquired on the
MS:MS_COMMENTS                   	Orbitrap mass analyzer. Full scans were acquired at a resolution of 70,000 at
MS:MS_COMMENTS                   	mass-to-charge ratio (m/z) 200 over the m/z range 70–1000 with the ESI
MS:MS_COMMENTS                   	conditions as follows: source heater = 350°C, sheath gas = 35 (arbitrary
MS:MS_COMMENTS                   	units), auxiliary gas = 10 (arbitrary units), capillary temperature 350°C, ion
MS:MS_COMMENTS                   	spray voltage = 3.0 kV (positive ion mode) and 2.5 kV (negative ion mode),
MS:MS_COMMENTS                   	S-lens 50%, and automatic gain control = 110¬¬¬-6. Tandem mass
MS:MS_COMMENTS                   	spectrometry experiments were performed at a resolution of 17,500 at m/z 200 on
MS:MS_COMMENTS                   	each sample with the higher energy collisional dissociation energy set at 20 eV.
MS:MS_COMMENTS                   	Data acquisition was carried out using Xcalibur software (Thermo Fisher
MS:MS_COMMENTS                   	Scientific). Before analysis, the Orbitrap was externally calibrated using
MS:MS_COMMENTS                   	ready-made calibration solutions (ESI-negative ion calibration and ESI-positive
MS:MS_COMMENTS                   	ion calibration solutions) obtained from Thermo Fisher Scientific.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area values
MS_METABOLITE_DATA_START
Samples	FH1_4	FH1_2	FH1_3	FH1_1	FH2_1	FH2_3	FH2_2	FH2_4	FH3_3	FH3_1	FH3_2	FH3_4	FH4_2	FH4_4	FH4_1	FH4_3	FH5_3	FH5_2	FH5_1	FH5_4	MH1_4	MH1_3	MH1_2	MH1_1	MH2_4	MH2_3	MH2_1	MH2_2	MH3_4	MH3_3	MH3_2	MH3_1	MH4_2	MH4_4	MH4_1	MH4_3	MH5_3	MH5_4	MH5_1	MH5_2
Factors	Sex:Female | Timepoint:4	Sex:Female | Timepoint:2	Sex:Female | Timepoint:3	Sex:Female | Timepoint:1	Sex:Female | Timepoint:1	Sex:Female | Timepoint:3	Sex:Female | Timepoint:2	Sex:Female | Timepoint:4	Sex:Female | Timepoint:3	Sex:Female | Timepoint:1	Sex:Female | Timepoint:2	Sex:Female | Timepoint:4	Sex:Female | Timepoint:2	Sex:Female | Timepoint:4	Sex:Female | Timepoint:1	Sex:Female | Timepoint:3	Sex:Female | Timepoint:3	Sex:Female | Timepoint:2	Sex:Female | Timepoint:1	Sex:Female | Timepoint:4	Sex:Male | Timepoint:4	Sex:Male | Timepoint:3	Sex:Male | Timepoint:2	Sex:Male | Timepoint:1	Sex:Male | Timepoint:4	Sex:Male | Timepoint:3	Sex:Male | Timepoint:1	Sex:Male | Timepoint:2	Sex:Male | Timepoint:4	Sex:Male | Timepoint:3	Sex:Male | Timepoint:2	Sex:Male | Timepoint:1	Sex:Male | Timepoint:2	Sex:Male | Timepoint:4	Sex:Male | Timepoint:1	Sex:Male | Timepoint:3	Sex:Male | Timepoint:3	Sex:Male | Timepoint:4	Sex:Male | Timepoint:1	Sex:Male | Timepoint:2
2-Piperidinone	8795.128993	2039326.935	903393.2681	77117.01303	77796.67631	25486.22275	45321.01537	20783.77311	23300.80248	103983.0123	29463.8451	29282.88698	1396424.444	1899252.769	37272.40145	3651510.09	157790.0043	124180.8581	106598.3303	32165.25321	2091409.55	3006041.339	3841024.401	6148789.429	686179.2998	979817.264	2443530.102	1139825.395	19919.43294	58631.13503	36927.42753	314945.2553	116209.2662	4561395.902	325915.1939	1422601.616	168955.9484	1119735.811	321050.6147	327552.611
Carnitine	198559.3094	19714037.68	9322184.204	16037994	8982140.533	7614541.88	10590142.03	11720064.68	6264608.944	9283154.863	8547937.46	17047462.88	18254675.83	7576091.144	7084392.736	9101492.631	5855013.536	13608112.24	12342500.01	4312556.639	6827489.015	4130754.812	8906270.062	5792588.71	6385264.659	5100024.875	5615968.906	5412106.927	3820187.143	4430180.391	4795328.321	6345756.586	2435859.28	4199467.732	2032131.117	4644265.941	2872351.101	5799532.86	2944248.22	2767422.584
Thymine	2625.922198	157078.3186	150315.6837	215787.663	147677.2109	140253.8323	160021.0967	151753.8188	135518.9561	169563.5386	156885.587	168007.7114	154277.8127	111905.9378	183360.5883	191431.5092	80450.87104	69449.67634	103671.3442	83567.28961	54979.76776	51693.66086	49926.18296	62845.33852	70641.04519	129233.8384	109271.0888	100113.6966	72683.49541	105350.9618	108056.0451	113093.3316	220003.7286	93748.67091	136132.5359	298879.7086	218839.0444	101503.04	178892.3615	191196.0702
Betaine	1103523.591	18105076.56	12879457.34	34046720.81	24798142	17724732.58	19613236.11	17242704.38	18364342.82	25257504.52	21198472.1	23890599.96	20183764.09	12700678.31	18224846.66	14085745.72	12223833.99	22083931.23	22971605.34	15155477.68	13657385.14	12570855.07	19527848.39	24452609.14	10518283.81	12057229.77	19487534.55	10443653.02	14921590.77	11945846.61	14385738.88	28739780.17	22237756.55	17884188.94	11551068.67	22603465.73	16789700.87	18537946.14	22683872.68	18944410.3
CAR 2:0	88273.7543	44998136.64	23280507.91	31285528.82	10737123.24	10259760.67	12593229.95	15729174.11	19449538.91	14954776.57	24815516.81	47837978.22	44070388.37	16378645.89	15517379.35	14913848.62	17199181.65	38725163.46	19692242.64	15001981.18	17833976.33	13131126.14	31056048.55	5062464.425	15797481.08	15577174.27	3375232.958	16242975.25	12412029.6	13674173.26	15636859.09	7938529.679	7814411.415	11309392.52	7109739.058	12058726.92	9698102.652	19665768.7	2039291.406	9989622.97
Kynurenic acid	2192.781402	143205.6404	116895.0244	609564.9286	13263.89299	159805.6346	23132.55585	282328.1221	88426.02986	139494.5424	106472.2368	86916.30665	89292.1166	30760.65596	365036.5288	55788.60348	146765.0756	111273.0974	189238.819	97675.16811	1554.444611	2422.83308	2767.46862	2326.655759	1553.405917	2123.677851	1864.906368	2338.91921	1887.813142	2246.70317	2466.450638	2295.257592	3249.87541	1867.976309	2759.503991	3255.62582	1493.907649	2369.379385	3693.992456	2894.80587
Tyrosine	18905.77749	16612935.73	13204989.22	11352971.22	4016673.738	3707219.003	6914760.28	7688684.563	10979097.63	10719110.43	12559815.96	18154736.36	17250043	9850543.45	4529350.416	7386900.774	12722380.16	22881419.35	16180968.21	9412299.4	9172636.919	6241396.067	15540838.85	2753.667578	4552731.564	5391359.947	3232.959851	7026797.853	6272786.127	6966075.878	7897914.371	45927.94586	8062282.727	15567673.71	5514033.569	7917459.139	5870417.452	13051637.16	3145.094104	7311448.428
Kynurenine	519.5669173	243168.4477	150611.847	484585.7998	29021.49624	50752.80979	17883.79361	291118.4923	134201.6039	202675.6592	125469.8966	106728.8569	203027.4472	71826.98093	165957.7498	78329.38158	161778.2781	180646.0429	187503.7159	122200.9169	12956.91285	19984.19653	14409.69777	25626.08961	11396.81663	9161.182754	23273.46938	13124.69269	13706.97708	22094.49195	17455.94383	18081.41393	10166.82546	13078.15075	6204.432735	8516.858922	10847.72701	7130.453139	4893.446419	8794.313811
Proline	36580.70525	75273882.33	32722445.09	108029217.4	52655952.17	21167320.82	51356634.21	38023172.76	35961280.3	73490408.05	40625721.01	55574837.35	50980133.51	22512794.15	30823269.58	31992609.37	57382882.36	101715659.9	138185428.9	42283571.16	58909169.64	48895667.41	91359271.82	67438248.78	36589423.35	35308458.23	30580488.57	39221478.23	33534808.5	41489595.94	42633688.24	51972656.25	40598615.5	54144218.01	41196716.3	57079705.93	47798633.57	75791163.99	20163870.11	49856895.19
Indolelactic acid	1324.088004	1032831.035	938131.3146	435611.6805	120434.8139	883115.3434	199044.1664	515112.142	812343.44	353245.796	777670.0965	1463531.82	457585.3973	653399.481	309278.5564	734287.0061	755484.9707	973617.4289	1046701.255	1271214.323	300008.4978	290094.3226	687720.2658	145922.6976	452994.2553	621319.9005	237139.2728	662810.2498	316456.1305	460398.5349	639516.2784	365205.7736	1058642.482	416507.7055	595318.9833	1007659.007	1017055.687	562398.5482	167940.5378	1432564.767
Nicotinic acid	8738.334387	99941.46546	186071.5844	392246.3617	114171.2756	104654.2642	140115.1803	177828.743	177271.0293	276908.4056	234100.6796	350391.7563	473546.1822	142887.8344	189822.9751	216662.2571	134531.6585	270468.4139	353895.2994	118226.7525	309968.4491	261881.7155	413900.4592	324957.1349	171010.766	219334.5664	224510.0048	171988.6681	192859.288	243604.5689	254940.1583	379896.9577	149422.4827	189266.1738	174720.8844	259471.2981	195732.7006	265227.6949	409420.774	181149.0927
N-Acetylmethionine	1308.260673	332641.3892	286480.115	223182.697	52737.15125	87184.0671	103824.3486	187755.9525	241333.1316	150980.9136	256342.0863	419508.4162	434567.7863	212647.4329	130944.9361	324224.0402	198237.8346	319987.743	152152.7119	168964.493	124429.9545	155196.8212	239642.6242	161248.8527	1467166.774	2938361.477	2952371.498	3387978.516	805524.9427	1191732.29	1305365.905	1455606.625	327818.3914	378655.9026	1418701.272	488379.536	1597741.022	766885.0043	393211.1159	1887483.528
Hydroxyproline	510696.812	14401434.48	12454937.39	12539644.82	4275078.549	2662554.879	4965774.928	5511273.587	9009087.023	10235282.68	9496989.143	9526476.562	13268057.88	7902523.751	6488524.927	8406025.516	6618093.208	8816204.73	10279145.1	4869244.63	7541860.748	5852528.692	8339903.951	3450150.295	5871648.857	5972301.22	4530369.932	6602856.484	7524842.828	8150036.337	8209193.744	4451775.526	6488406.743	8067527.308	3854922.42	8288217.366	5088863.047	7806073.311	2241734.35	4656401.123
Guanosine	1745.057077	6528371.124	4543523.077	4650740.612	3045142.419	2271947.827	3535689.779	3344852.875	4252885.972	6530105.868	5566655.175	5401678.437	5466101.039	2725687.303	3577807.471	3422089.534	4382157.175	8556714.886	9363395.1	1730863.144	1613444.154	1548871.635	2568739.481	2028534.557	1249865.094	1714559.507	1974679.755	1712683.449	1108973.771	1766839.208	1849402.544	2305253.797	997298.6122	1371009.106	1132754.512	1687372.737	1424263.275	2109001.91	2044813.699	1508672.886
Guanine	3775.020328	4406344.04	3061052.152	3130077.163	1962406.49	1374559.858	2366780.862	2214000.896	2756345.25	4380309.628	3779727.098	3652729.323	3711420.904	1768818.355	2349293.221	2280158.392	2928386.464	5896365.418	6472912.413	1117152.848	1122095.467	998675.3623	1705468.554	1023382.806	803871.7222	1072391.122	1235163.192	1100295.93	717079.7041	1131212.226	1170486.622	1282823.972	637063.5594	896700.2165	717384.7121	1103073.856	918564.0269	1385906.478	1211853.925	967961.4468
Glutamine	147613.7926	11678374.33	7538886.297	12453929.94	5447764.485	3038014.576	5503162.015	5628053.854	6836773.597	8300103.869	5709979.537	9139316.068	9624493.877	4665641.412	5863414.685	5127691.054	6568819.438	10744175.01	12581945.7	6103872.31	7018571.041	5076642.04	8002088.252	3748886.911	5189838.039	4659035.278	2885049.115	5421841.414	5636085.93	5919210.722	5541233.256	3145642.002	6474451.358	6342127.22	3504079.123	8187100.761	4467654.241	8274610.725	2983406.165	4010262.945
Inosine	3126.48604	5694607.552	3882289.462	4281650.551	2456781.194	1622915.883	2166462.765	3154682.666	3846198.117	5526825.623	5630450.335	3140068.352	4237301.852	2448289.859	2830071.451	3506550.466	4330297.19	7420536.146	7288116.986	2302489.704	2203125.507	1979378.117	2714008.895	2356686.738	1386004.757	1472211.696	2521797.81	1290589.488	1323020.913	1931841.106	2025908.673	2939615.31	1387724.459	1723089.193	1273496.717	1507959.923	1606089.701	2008731.089	2285613.714	1493159.987
Adenosine	1247.261427	194985.7342	6793.533116	282616.4241	771905.1587	65576.91586	509043.7034	135436.6107	74690.24127	214244.1755	169073.836	291693.798	176055.5903	131786.7125	230179.3199	132926.0358	128698.4399	378727.8029	378383.3061	101726.2469	153297.6902	77435.06606	343812.399	409227.9484	59569.13866	73839.86895	323171.2788	100102.3758	102739.549	4287.721077	107789.8075	367867.7429	86690.24869	75055.36388	138197.4279	50612.43736	57296.25862	228912.1069	389866.0662	148680.7654
Urocanic acid	12397.89811	42865775	27662979.11	43746535.93	12190420.07	8285835.171	19181810.65	16143239.37	16688582.77	15280743.77	16714969.02	52392677.61	45063290.88	12909848.23	13283669.76	18501610.13	24507026.85	51105910.84	35414659.96	21006869.14	41664289.85	27255703.21	64122572.4	7925466.082	37941434.64	44346376.74	17082000.37	56880638.9	40338147.47	63242187.75	72757169.97	47231272.5	64186115.69	57195147.62	40472113.09	109120150.8	63465857.7	63371217.04	15829313.73	54336935.79
7-Methylguanosine	276.9238175	1710.434383	976.7969633	2079.423545	1037.904857	616.8995618	1290.077482	974.5756862	1074.567303	1715.332198	1503.771786	2185.478856	1856.621738	1140.101145	982.8015117	782.2549274	1530.381078	2000.946567	1814.062826	1026.472979	879.7065923	765.3967692	2205.641234	40127.96115	1335.791996	972.5038754	51317.51382	909.4903396	815.6065703	1552.476569	1548.354666	80676.36615	1311.86009	1236.251648	778.834231	1867.415921	1499.746095	1828.399855	21885.75013	1286.909684
Uric Acid	9599.548371	13906676.19	7374564.069	5344020.101	542001.3817	460037.9013	1219160.456	3694153.586	3382883.162	6485930.298	7852261.444	2830419.784	7961784.123	5105796.076	1431092.208	5639011.393	5546309.756	15494587.91	13625528.03	1239820.594	4101513.425	2089959.326	6581741.499	2917645.838	1385392.914	1279271.976	2415165.296	1064839.962	1283033.027	1836797.704	2210230.64	5053345.987	976662.1389	981036.1297	876766.9212	3758545.553	1903435.844	1593455.944	2913736.256	1601696.945
Glutathione	366.4948549	11145.32601	4156.085346	6159.989228	1997.340004	2313.770991	12916.20178	13325.05419	3742.963492	3885.314453	23001.72603	8148.115462	9834.586322	65356.88492	2345.579495	3037.536784	4583.908672	11786.80733	8061.475945	276399.5708	9198.541339	3677.254451	11904.93169	3624.442751	112732.3098	5978.192116	4147.821481	6847.352755	15340.7833	8866.069993	9411.628415	6565.671129	7741.960245	6532.570248	14798.75667	13886.58266	12029.46743	11410.6152	2599.735374	26811.71122
Indoleacetic acid	715.0125757	10314.05633	6452.305481	5449.890604	6012.285757	10797.339	4305.093859	8191.18804	6268.987346	5328.935012	11802.86116	20994.29504	15136.76618	8060.233312	3415.873511	9471.631129	7408.202591	9606.185336	10102.52806	24806.88105	9972.958341	9659.670272	27978.57871	7856.905425	20396.9199	26784.64674	13620.38792	35941.96927	6384.045177	10890.72057	17165.10863	26750.41997	63498.48966	85542.59769	34144.80542	99383.00774	45873.49446	24815.90861	14623.12289	65024.49178
Acetylcholine	24695.44816	9008090.613	3757702.261	9143338.064	6085998.827	3756800.721	6120512.383	5538830.459	3768963.133	6746848.759	5843259.359	9957821.215	9864307.122	3560384.757	3837069.204	4070328.498	2653276.391	8942582.093	8894796.049	1505318.623	4150128.065	2210621.659	6974644.929	3744307.034	3858213.852	2926095.947	3505423.39	3580356.493	1097509.958	1063337.705	1572631.442	2829678.985	871794.8132	1494405.342	978892.6476	1600243.989	1173593.942	3366684.014	643979.9548	1869085.22
Pyroglutamic acid	101307.8691	6333084.34	4085204.505	6343553.163	2868822.744	1584858.497	2757149.649	3060653.27	3424217.211	4244351.802	2947628.718	5042876.687	5275092.42	2519538.337	3091291.886	2670425.095	3538292.514	6138556.614	6620901.022	3290088.171	4582642.993	2915938.489	5193893.684	1537923.268	3582654.37	3105780.525	1709840.613	3591213.348	3712454.39	3715912.663	3933462.193	3147362.942	3632824.78	3820069.361	2487237.221	4821730.737	3054531.596	6383093.028	1459253.274	3182747.238
Phenylalanine	68816.45667	40019734.09	36244549.14	43016509.04	16310823.79	11701481.43	21855935.13	18108155.35	30438850.05	35503434.43	31059707.7	36990087	33846236.52	22540800.76	19467166.42	21759321.65	33259009.66	46756426.97	50203547.8	26491319.72	19389881.95	15349219.04	26470354.19	23330345.29	13141010.16	19021604.42	30163330.12	24279238.92	25095623.72	30069130.98	30462007.31	39217688.45	21496116.62	28741235.41	18793022.23	25276801.54	20947259.8	25632760.29	34409990.87	26159317.39
D-Tryptophan	32257.67393	11576660.54	12072101.47	13788157.78	12378427	9915972.71	12223207.84	8765397.625	11429109.79	10120438.05	12956762.11	9161311.096	6415542.292	7528964.658	8821280.497	7124336.643	12303697.2	12034948	12721356.07	10634428.55	5630960.539	6031740.8	6344552.642	4166754.144	6671451.292	9491768.845	6411587.143	9380166.157	7597776.425	10269721.85	12693494.65	4770507.859	10703222.64	6936482.232	11659123.79	11074540.87	10611982.76	7470240.775	2040345.655	12631092.46
Niacinamide	7223.158368	71594.30233	14352.1409	67980.70064	17854.14602	13877.61075	37129.7417	15360.02563	10234.81917	26562.81629	17341.54682	48365.76503	68647.87971	13881.02358	10047.44706	11752.6612	11977.9266	83942.02615	44254.32472	15660.46848	25554.59855	8037.293137	57833.96763	72315.93614	8987.302462	7104.950426	53371.97383	36766.87401	6001.755855	9714.726524	18682.64068	281883.6515	25808.38835	12490.67644	5213.907617	37338.399	12581.22016	35937.78648	161084.9909	10956.48093
Aminosalicylic acid	2593.006059	38077.43884	35188.65659	928886.4756	1356787.69	858080.4853	389814.1998	1157639.654	1309363.016	581844.6252	1811351.152	1365653.839	29369.29994	579071.5555	1116137.848	513351.0618	494971.8344	47971.77374	668648.8733	471868.2463	662453.4372	471972.656	79064.18616	658928.7196	1071017.362	783098.7686	1568806.661	982928.9386	1710795.578	132225.4259	2655961.585	1469795.817	1441399.428	1644628.265	1300309.017	216558.7039	2237782.338	1171241.061	2898947.42	2088947.933
Hypoxanthine	1098.697322	38972274.83	24764691.74	41345611.44	10986277.68	7079264.048	18231197.8	14601839.52	15080390.97	14546485.83	15136747.64	47478952.98	40969056.12	12280715.6	12124093.39	17365240.96	22486230.78	46785237.04	32297320.57	19271381.85	39298546.48	24517181.36	60521805	7538823.444	36610703.05	39721654.18	16668895.02	52958756.43	37867655.34	66334824.43	72206499.61	43968518.77	63593551.25	52681911.07	37326501.83	107768939.1	62627313.33	61178964.1	15405318.96	49989597.84
Histidine	26608.74461	895368.3405	800901.4546	918514.4419	457361.9019	272014.0995	521301.6919	457820.5372	823429.39	739424.4475	717203.1646	594060.6337	736998.3737	669359.1779	428283.5999	460801.66	804147.8867	757041.1184	853574.794	661777.5599	377271.402	279729.652	352025.3857	372763.957	190253.7767	254304.2709	419360.2469	321527.2925	245893.0819	381447.8667	354486.7964	469552.6335	503333.3979	571844.818	282213.0265	623377.7401	383742.237	353352.5764	557108.8568	274740.3342
Pantothenic acid	7987.84987	625394.3387	239165.0169	656590.6936	226477.8436	112882.1913	118156.6311	76212.40049	189252.4632	249112.566	244865.3785	331054.7722	534093.7127	131119.3566	217930.445	202355.0196	145153.3937	947397.8799	737333.3139	105248.1642	104688.914	93705.94182	271561.6902	209438.7791	79791.71566	77604.50479	206552.6759	127943.7779	85629.37244	118030.7666	143158.6258	476638.2636	186733.9199	107462.0518	126890.1903	250721.0581	95258.66604	218900.9346	517433.1832	156977.2002
Cytosine	3183.345735	2973943.983	1582510.784	1467660.938	925836.544	420386.0842	754085.4363	714564.793	563328.5658	1965408.031	2561231.483	1356117.213	2008505.738	879451.1361	690147.5156	1230621.581	925849.772	2750419.875	2990374.858	843074.0559	908530.6457	841701.6011	1502169.458	118610.6342	840196.1982	482413.4128	133544.7278	474614.0663	316183.9015	200574.7079	349342.4316	228725.5358	507747.9816	676837.2148	136409.233	414096.2329	202982.1009	466403.1379	109859.2957	448553.0641
Cytidine	1047.162861	3415447.702	1861140.686	2189427.719	1036997.999	545439.2781	1132580.783	768846.2542	866299.0986	2739150.078	4026972.002	1448518.229	1834137.618	1211707.045	940665.706	1585121.84	1450072.41	3133611.695	3485384.677	1241515.491	1271399.932	1116486.657	1771072.439	190101.8379	1301528.961	766757.7296	269292.7861	703206.3124	316244.8745	381560.9388	660883.6277	763206.8184	936478.9496	1006261.665	275370.5159	509731.5505	349486.4761	661093.4099	398574.0212	804621.2836
Arginine	45599.94742	8819753.324	5835041.539	5694278.216	1860619.575	4551456.222	4031453.258	4601669.861	6150678.986	1738726.507	7646531.244	8029213.167	9485794.179	4589529.634	1572020.153	7685444.996	4953364.575	9003167.474	3755248.423	4422366.364	5076873.447	5430504.573	7531342.417	1966936.358	3297240.18	3121318.727	2385910.246	3464282.017	2262222.297	3713790.85	2907955.562	2843906.046	7162484.059	7728678.632	2712289.63	8292629.782	5011773.59	7872573.026	5321707.975	5450334.652
2-Hydroxycinnamic acid	2907.657423	3296458.803	2567440.958	2330843.914	808174.1996	744287.9881	1384661.846	1567971.988	2186620.561	2085017.536	2426032.594	3604286.472	3409599.927	1903314.46	916512.1395	1392665.205	2475854.022	4428508.15	3253077.083	1831773.637	1782299.695	1229801.653	2946471.43	1846.017437	896859.4392	1060144.724	1216.404064	1349277.124	1172664.412	1394092.402	1573661.998	8341.821568	1551366.276	3020781.514	1060967.135	1548025.688	1168035.948	2486087.188	336.7472647	1440707.825
Phthalic acid	1708.078085	84769.75473	51778.06736	140301.9228	32470.62167	61743.46607	73893.87407	52590.79871	59423.30977	144449.5748	71111.78136	80185.75443	82148.63009	52606.89959	49988.44512	44370.15051	87697.12893	116232.3703	119512.6033	33385.76117	131612.3356	81882.64446	142627.9089	220765.7088	40303.76014	71776.52497	153262.9473	106479	76521.95653	138272.2521	160368.1361	321002.688	66262.48873	53142.34362	115846.147	89353.96171	110953.4511	92945.51634	59441.64979	120281.0631
CAR 3:0	3740.410421	6945173.785	3765374.189	7188643.717	2616265.989	3642331.458	3854482.895	6120248.636	2699095.581	4122300.895	4745701.656	6879766.717	11587951.85	3565146.447	3880817.314	4552902.455	1380858.755	5309163.225	2849884.524	1156305.956	1344155.736	950956.9622	1967455.196	1598577.171	1280425.941	806968.4672	1294368.736	754566.6136	877309.8002	297917.6769	398441.7776	1025877.743	597234.5466	711710.0096	166716.5573	592224.214	286005.5286	1337151.309	745472.4616	635974.5615
CAR 10:0	2503.804366	138518.9421	73263.66192	128795.0742	94036.85302	65241.99001	85439.70071	114576.8439	110704.5741	127742.5021	133138.4317	113066.4574	126534.9234	90012.01197	86481.76894	80313.42084	64918.74833	109993.7911	63921.45842	54390.82121	47289.14045	38216.44499	53480.03419	50846.81167	49661.57656	45039.92841	51130.40228	46440.10848	45867.05072	26157.98697	37318.26459	39152.10499	30798.05596	36220.44274	22606.78516	30079.42227	26619.66347	33499.21828	40983.83222	39020.92995
Tyrosol	4575.934158	53031.04703	22442.45534	33682.26147	38842.76189	55755.44819	61119.7183	19029.25817	29058.47104	29593.98332	28921.86869	42712.21702	61613.67294	38960.46952	31694.53767	22661.618	15810.55106	75857.44024	81211.68318	31407.42049	23259.04089	42804.59129	54655.98186	38946.63983	35519.96936	35580.12272	36042.09995	22613.51514	42621.58238	22063.3504	22775.38518	29323.04657	21298.48203	19296.47238	30883.72606	24301.35711	14966.41831	24233.88469	70039.19323	19134.19949
N-Acetylaspartic acid	605.5250408	179582.5717	23389.6822	72708.50709	16298.37442	11901.68271	18982.7495	23352.41159	11451.21157	15999.35146	26690.67819	150326.6896	135447.7524	16725.62358	9660.060448	20818.00512	22228.08835	152471.7071	182629.0414	25352.22541	51919.07846	8124.752403	108268.8675	43668.06152	8308.990485	9880.238324	15076.09215	39441.60764	15266.13732	14677.50959	15587.60887	75966.56199	19717.93316	25826.51501	10633.74013	46990.24707	14372.09301	50157.21438	16556.72543	13256.96715
Spermidine	1110.444774	305310.9168	71601.6886	246500.6014	148881.2612	46374.34255	86467.28993	126361.5628	115384.1933	263007.1598	174651.967	199093.5768	329259.8285	136190.8736	82614.5097	137846.4266	72009.50209	250270.1787	318347.0027	54936.346	282020.6307	120670.2159	687067.1208	511528.1182	74067.14109	79734.7332	140326.5818	112720.1325	133629.3742	132059.1013	315548.2963	446310.2561	171007.532	242933.6523	95890.32925	433535.3468	222058.3354	502250.9219	97325.47322	309466.6783
Norspermidine	1318.417998	499599.1021	76159.94402	404056.2857	111988.2977	46870.09173	123080.4274	115296.7178	81692.81272	181483.6769	233520.759	354114.0536	343928.1261	115509.3164	83822.54533	69642.10407	86128.90562	787704.7897	849294.5076	43303.84478	140469.9208	44792.85534	420213.8924	280097.6446	43682.69317	50189.81759	132700.8902	60617.52511	87521.58872	104736.6546	175202.9474	496136.857	61504.88572	100263.0814	54719.25247	223061.1511	126637.1954	379478.4443	85018.08066	153316.9569
Oxidised glutathione	588.9315873	43121.80215	6852.077666	15620.97501	1807.756077	4650.200937	6064.02329	12324.20504	2685.304343	2604.473142	2627.438828	61424.64957	50724.46423	4187.189909	1607.281736	4548.650068	4267.025556	97362.05626	13988.11276	7059.467578	13005.38836	4307.149968	61611.07543	998002.9107	1947.626073	669.1956599	123040.4854	801.8372947	2212.927123	1401.886942	10729.6911	1183504.984	11413.03537	4687.531091	3502.839337	12570.93087	5493.506929	40727.08114	80005.19622	9154.339886
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	PubChem ID	Formula	retention time	MW
2-Piperidinone	-	12665	C5 H9 N O	3.038	99.06876
Carnitine	C00318	10917	C7 H15 N O3	0.886	161.10516
Thymine	C00178	1135	C5 H6 N2 O2	2.97	126.04304
Betaine	C00719	248	C5 H11 N O2	0.866	117.07913
CAR 2:0	C002571	7045767	C9 H17 N O4	1.293	203.11579
Kynurenic acid	C01717	3845	C10 H7 N O3	3.226	189.04259
Tyrosine	C00082	6057	C9 H11 N O3	1.668	181.07394
Kynurenine	C00328	161166	C10 H12 N2 O3	2.96	208.08485
Proline	C00763	8988	C5 H9 N O2	0.911	115.0635
Indolelactic acid	C02043	676157	C11 H11 N O3	3.675	205.07392
Nicotinic acid	C00253	938	C6 H5 N O2	1.275	123.03214
N-Acetylmethionine	C02712	-	C7 H13 N O3 S	3.271	191.06169
Hydroxyproline	C01157	5810	C5 H9 N O3	0.865	131.05826
Guanosine	C00387	6802	C10 H13 N5 O5	2.252	283.0921
Guanine	C00242	764	C5 H5 N5 O	2.252	151.04941
Glutamine	C00064	5961	C5 H10 N2 O3	0.864	146.06909
Inosine	C00294	6021	C10 H12 N4 O5	2.23	268.08091
Adenosine	C00212	60961	C10 H13 N5 O4	2.185	267.09675
Urocanic acid	C00785	736715	C6 H6 N2 O2	1.275	138.0429
7-Methylguanosine	-	445404	C11 H15 N5 O5	2.075	297.10708
Uric Acid	C00366	1175	C5 H4 N4 O3	1.271	168.02831
Glutathione	C00051	124886	C10 H17 N3 O6 S	1.273	307.08357
Indoleacetic acid	C00954	802	C10 H9 N O2	3.841	175.06335
Acetylcholine	C01996	187	C7 H15 N O2	0.945	145.11024
Pyroglutamic acid	C01879	7405	C5 H7 N O3	0.875	129.04268
Phenylalanine	C00079	6140	C9 H11 N O2	2.993	165.07898
D-Tryptophan	C00525	9060	C11 H12 N2 O2	3.197	204.09002
Niacinamide	C00153	936	C6 H6 N2 O	1.361	122.04815
Aminosalicylic acid	-	4075	C7 H7 N O3	3.134	153.04252
Hypoxanthine	C00262	790	C5 H4 N4 O	1.276	136.03848
Histidine	C00135	6274	C6 H9 N3 O2	0.833	155.0694
Pantothenic acid	C00864	6613	C9 H17 N O5	3.049	219.11058
Cytosine	C00380	597	C4 H5 N3 O	1.205	111.04354
Cytidine	C00475	6175	C9 H13 N3 O5	1.206	243.08548
Arginine	C00062	6322	C6 H14 N4 O2	0.831	174.11168
2-Hydroxycinnamic acid	C01772	637540	C9 H8 O3	1.668	164.04741
Phthalic acid	C01606	1017	C8 H6 O4	3.758	148.01608
CAR 3:0	C03017	18824	C10 H19 N O4	2.531	217.13147
CAR 10:0	-	10245190	C17 H33 N O4	5.03	315.24089
Tyrosol	C06044	10393	C8 H10 O2	3.299	138.06811
N-Acetylaspartic acid	C01042	65065	C6 H9 N O5	1.265	175.04798
Spermidine	C00315	1102	C7 H19 N3	0.71	145.15787
Norspermidine	C03375	5942	C6 H17 N3	0.705	131.14226
Oxidised glutathione	C00127	65359	C20 H32 N6 O12 S2	1.772	612.15294
METABOLITES_END
#END