#METABOLOMICS WORKBENCH EmilyLette_20210225_014138 DATATRACK_ID:2509 STUDY_ID:ST001721 ANALYSIS_ID:AN002805 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, 1g/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 #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE 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 = 110¬¬¬-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 Uridine-5'-phosphoric acid disodium salt 31904.42859 177303.5079 26361.21716 336286.9114 284496.3138 51048.16339 46580.30549 544304.5351 40708.82587 276840.9992 60211.92045 178431.8637 80265.81777 123087.904 662435.2612 61917.42375 32907.76447 84462.2479 52855.27723 126758.6482 120473.5737 32011.254 71792.17373 51506.75842 47830.67427 14942.65662 19903.12391 35811.06605 39232.92642 5912.837354 169170.8346 49497.26362 688093.1342 151691.9516 22169.70851 107096.3726 33102.1552 287940.4999 9056.474022 52546.81216 Sucrose 70542.41055 72891.59562 69522.03939 52707.02607 117908.4841 73839.19037 99984.1763 39624.85158 13019.74452 100574.1753 94619.24022 33875.56939 60536.05699 47711.80617 113732.0642 60697.18232 29543.3592 179457.6336 97747.55846 21999.78918 85905.62826 107926.0567 87487.0463 135799.9351 83096.99953 53168.3698 142680.7573 88442.02317 29795.0413 11639.84466 3064659.87 231467.5968 52908.12418 31299.65072 49445.3477 38308.05557 12946.44137 28787.55149 302144.9727 74325.7096 Azelaic acid 1395797.305 9657471.261 1260963.175 5336086.544 12724355.41 1511973.787 5195489.553 1292388.105 1419078.654 10010106.17 4646533.428 1256484.845 5496863.146 1260821.045 24631277.86 1230609.482 1199759.247 5129309.61 5680334.324 1275736.018 1963290.962 1154036.585 4485841.111 8456793.791 1197521.237 1231666.377 9671545.192 5643126.422 1367300.45 1691146.148 10210249.18 13525880.18 20449485.37 1231364.265 3465165.3 1472379.862 1226012.694 1327562.092 9564634.412 4531201.482 Kynurenic acid 1412.649885 67879.04057 42307.0614 272875.6308 7839.562664 62413.53934 9306.409631 220382.7761 31153.09686 81662.74444 43405.68369 40690.18242 39589.20033 20068.79426 283518.1989 24734.42336 54130.30884 54399.19579 59549.30683 50601.61359 1763.659907 1000.168875 1198.857694 1092.478308 1113.985072 787.9857902 1223.943601 1592.868276 900.0751328 694.571393 1197.643463 2128.592862 1792.734393 1202.888475 933.8487814 1199.962065 1370.800498 2585.273247 998.5005161 1197.629382 Inosine 4445794.114 5823097.752 5061786.269 5553904.456 3710286.25 1632924.827 2367736.45 5964960.003 4730225.797 8876111.812 7225637.319 2529044.777 3431409.099 3541887.147 5690405.792 4291729.281 5000151.457 6447830.914 8052726.657 2956817.552 2860025.839 2152064.282 1945883.103 1231189.449 1409277.907 1567598.787 2160913.256 1317561.472 1316459.112 1997641.309 2532323.794 3215983.233 2403431.529 2291247.745 862715.3885 1601828.579 1551048.869 2045035.43 1676359.296 1439277.279 Deoxyinosine 29229.02048 51230.64886 45241.78978 69351.97166 61521.9116 62539.63569 65434.21054 89601.39565 34051.2439 46692.15091 39368.66988 36596.74328 33147.94788 32506.14178 68765.78043 34613.15241 43911.88485 45262.0752 49850.34782 45079.34136 26722.3581 27247.02534 23116.93987 183.3640152 22070.21044 44099.40894 233.1306524 32757.47376 24495.9952 37094.32092 44549.79035 6790.274531 40795.0211 39364.82842 38127.51102 91721.51013 67568.03704 39148.83608 434.3411672 76374.87543 Indolelactic acid 2506215.587 2303722.732 2371394.091 1124355.246 369964.2857 2222437.128 546768.2139 1850711.828 1997955.966 1074470.967 1775494.707 2939394.652 863910.9982 2108745.809 1278282.679 2003567.991 1832008.777 1831705.102 2071040.752 4075723.858 899603.357 784418.8431 1317098.753 333647.2487 1376940.865 1863025.278 785081.7371 1677802.5 895202.2405 1272460.452 1889425.399 1312825.017 3933571.725 1307195.794 1192497.858 2765298.783 2576262.229 1467643.574 761208.7291 3535838.386 Hydroxyphenyllactic acid 1055532.868 174708.8859 200822.9535 59940.2396 35152.10484 120422.3165 55979.84566 114395.4999 144434.0706 75803.00979 232544.2316 169294.6597 103849.5311 161351.2396 119515.411 119517.8161 135786.0778 125401.1642 77229.11595 196957.2466 141763.4975 92751.86393 114146.8743 70307.01072 100948.4531 83659.69385 74481.96498 94987.34734 69150.77051 71469.6286 96744.15568 60041.02824 95319.61365 276178.8398 32995.21649 139214.3342 93950.90583 114245.1463 108873.3111 109155.9165 Glutathione 1628.986326 8756.251135 1101.62743 1353.975015 10831.91156 758.5223982 3029.16889 1694.53477 10463.55038 1389.875999 3394.757056 8523.716705 1757.318208 1205.698405 15624.07122 909.1266002 864.5271025 2047.262666 16824.81048 96761.46405 1366.563527 785.3244392 2425.216067 693.4328332 31012.58036 702.6894447 14797.8397 17186.72603 4643.117283 727.5474882 1380.294491 13734.22742 1854.450862 1665.762191 839.9984648 5606.112815 2165.93514 3112.187641 973.3094181 7071.705797 Oxidized glutathione 8519.589502 115317.7517 19442.54602 120329.0035 127249.7395 38467.46771 53903.31594 747685.8613 17710.04164 86061.93716 17288.98418 111399.5792 63966.66636 183050.4377 381011.1918 53811.05015 12141.55057 68211.94773 5834.710867 98526.98655 224228.7786 26262.09787 56735.5731 112181.1129 27556.11734 5630.204428 99224.27724 12898.11819 8601.571482 2406.017304 118880.2286 206477.9062 890220.9019 139121.9363 6812.365099 65072.33191 25564.84753 277411.4022 139356.5346 46635.59505 Uridine 10730038.63 21874090.18 19678525.19 24904847.2 24221557.88 21090186.39 26799358.24 21769387.57 22935620.15 32561970.07 32443012.8 16631237.68 17142485.15 19272245.66 26476685.59 19253457.93 19526136.2 21276543.21 28529021.76 13967570.83 22704421.87 24155295 23151974.46 25714817.68 21454870.6 23473747.34 26263471.98 24078097.86 17154869.51 22414933.35 24395115.39 25740362.49 22582782.13 21705807.11 11222104.77 22470688.34 19889693.83 19298681.48 18961019.63 19260514.88 Tryptophan 70751.49963 132771.0561 119605.1682 172242.3194 179166.0394 109287.0062 149196.9799 93741.15531 124074.7239 116444.608 138680.0307 102241.5253 75308.34176 91824.42466 153989.4586 87095.70428 137005.4642 176239.1651 129331.9207 157648.3429 58074.37582 70469.44486 70766.73397 38016.62349 81449.1882 115532.6712 64449.5441 111109.6489 101722.9574 121071.5028 158625.3789 85257.11796 113136.7688 74032.37438 86119.10493 121025.6495 124131.9781 84658.65044 31419.11163 138633.5929 Pantothenic acid 7766977.348 222590.7265 159356.0732 440156.9869 394466.4805 97573.13457 90666.92641 165719.5249 132541.5686 366328.8019 167159.5079 101483.5044 116037.7544 184613.676 667947.6509 169517.4988 96878.62457 179077.2661 204277.1199 120529.842 132378.6966 74537.32322 63373.1696 149714.1454 69812.6577 60493.98985 192640.751 90050.1821 68362.48234 86152.31147 149777.6336 391808.6267 517797.5831 148780.2037 68228.14134 169089.9593 68934.27276 145959.0208 640716.5235 118502.5943 Citric acid 547720.8911 224977.9216 52242.06618 749659.0696 365190.0129 30394.43001 33725.66684 674937.402 60277.49188 405615.6623 148952.8901 104382.1512 77900.1136 258485.4333 563246.5728 58598.94747 27328.85698 607672.7408 66647.55593 120024.2698 1065159.625 212602.6602 131062.6229 325342.8637 53977.34512 11629.62069 220883.9055 52817.96656 31945.83544 15970.55784 258518.1156 322441.747 1152583.826 298013.909 18289.47868 195710.9815 26087.91456 200392.5479 157766.1975 102598.9385 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID PubChem ID Formula Retention time MW Uridine-5'-phosphoric acid disodium salt C00105 6030 C9 H13 N2 O9 P 0.951 324.03573 Sucrose C00089 5988 C12 H22 O11 0.945 342.1158 Azelaic acid C08261 2266 C9 H16 O4 3.736 188.10403 Kynurenic acid C01717 3845 C10 H7 N O3 3.223 189.04183 Inosine C00294 6021 C10 H12 N4 O5 2.252 268.08085 Deoxyinosine C05512 65058 C10 H12 N4 O4 2.758 252.08565 Indolelactic acid C02043 676157 C11 H11 N O3 3.674 205.07327 Hydroxyphenyllactic acid C03672 9378 C9 H10 O4 3.188 182.05708 Glutathione C00051 124886 C10 H17 N3 O6 S 1.287 307.08369 Oxidized glutathione C00127 65359 C20 H32 N6 O12 S2 1.624 612.15131 Uridine C00299 6029 C9 H12 N2 O6 1.541 244.06926 Tryptophan C00078 6305 C11 H12 N2 O2 3.189 204.08927 Pantothenic acid C00864 6613 C9 H17 N O5 3.045 219.11021 Citric acid C00158 311 C6 H8 O7 1.289 192.02613 METABOLITES_END #END