#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