#METABOLOMICS WORKBENCH blumem_20220107_004533 DATATRACK_ID:3026 STUDY_ID:ST002053 ANALYSIS_ID:AN003343 PROJECT_ID:PR001297 VERSION 1 CREATED_ON January 7, 2022, 8:41 am #PROJECT PR:PROJECT_TITLE Resistance to NaFAc of in vitro maturated Toxoplasma gondii bradyzoites in human PR:PROJECT_TITLE myotubes PR:PROJECT_SUMMARY The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue PR:PROJECT_SUMMARY cysts that cause chronic and drug-tolerant infections. Here, we developed a PR:PROJECT_SUMMARY human myotube-based in vitro culture model of functionally mature tissue cysts. PR:PROJECT_SUMMARY In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts PR:PROJECT_SUMMARY tolerate exposure to the aconitase inhibitor sodium fluoroacetate. These data PR:PROJECT_SUMMARY characterize bradyzoites and tachyzoites treated with NaFAc. PR:INSTITUTE Robert Koch-Institut PR:LAST_NAME Blume PR:FIRST_NAME Martin PR:ADDRESS Seestr. 10, Berlin, Berlin, 13353, Germany PR:EMAIL blumem@rki.de PR:PHONE +49 30 18754 2572 #STUDY ST:STUDY_TITLE Resistance to NaFAc of in vitro maturated Toxoplasma gondii bradyzoites in human ST:STUDY_TITLE myotubes. ST:STUDY_SUMMARY The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue ST:STUDY_SUMMARY cysts that cause chronic and drug-tolerant infections. Here, we developed a ST:STUDY_SUMMARY human myotube-based in vitro culture model of functionally mature tissue cysts. ST:STUDY_SUMMARY In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts ST:STUDY_SUMMARY tolerate exposure to the aconitase inhibitor sodium fluoroacetate. These data ST:STUDY_SUMMARY characterize bradyzoites and tachyzoites treated with NaFAc. ST:INSTITUTE Robert Koch-Institut ST:LAST_NAME Blume ST:FIRST_NAME Martin ST:ADDRESS Seestr. 10, Berlin, Berlin, 13353, Germany ST:EMAIL blumem@rki.de ST:PHONE +49 30 18754 2572 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Toxoplasma gondii SU:TAXONOMY_ID 5811 #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 - PP_invitrocysts_mock_1 Factor:PP_invitrocysts_mock RAW_FILE_NAME=PP_invitrocysts_mock_1 SUBJECT_SAMPLE_FACTORS - PP_invitrocysts_mock_2 Factor:PP_invitrocysts_mock RAW_FILE_NAME=PP_invitrocysts_mock_2 SUBJECT_SAMPLE_FACTORS - PP_invitrocysts_mock_3 Factor:PP_invitrocysts_mock RAW_FILE_NAME=PP_invitrocysts_mock_3 SUBJECT_SAMPLE_FACTORS - PP_invitrocysts_mock_4 Factor:PP_invitrocysts_mock RAW_FILE_NAME=PP_invitrocysts_mock_4 SUBJECT_SAMPLE_FACTORS - PP_tachys_mock_1 Factor:PP_tachys_mock RAW_FILE_NAME=PP_tachys_mock_1 SUBJECT_SAMPLE_FACTORS - PP_tachys_mock_2 Factor:PP_tachys_mock RAW_FILE_NAME=PP_tachys_mock_2 SUBJECT_SAMPLE_FACTORS - PP_tachys_mock_3 Factor:PP_tachys_mock RAW_FILE_NAME=PP_tachys_mock_3 SUBJECT_SAMPLE_FACTORS - PP_tachys_mock_4 Factor:PP_tachys_mock RAW_FILE_NAME=PP_tachys_mock_4 SUBJECT_SAMPLE_FACTORS - PP_tachys_mock_5 Factor:PP_tachys_mock_5 RAW_FILE_NAME=PP_tachys_mock_5 SUBJECT_SAMPLE_FACTORS - PP_uninf_mock_1 Factor:PP_uninf_mock RAW_FILE_NAME=PP_uninf_mock_1 SUBJECT_SAMPLE_FACTORS - PP_uninf_mock_2 Factor:PP_uninf_mock RAW_FILE_NAME=PP_uninf_mock_2 SUBJECT_SAMPLE_FACTORS - PP_Blank_1 Factor:PP_Blank RAW_FILE_NAME=PP_Blank_1 SUBJECT_SAMPLE_FACTORS - PP_Blank_2 Factor:PP_Blank RAW_FILE_NAME=PP_Blank_2 SUBJECT_SAMPLE_FACTORS - PP_Blank_3 Factor:PP_Blank RAW_FILE_NAME=PP_Blank_3 SUBJECT_SAMPLE_FACTORS - PP_invitrocysts_NaFAc_1 Factor:PP_invitrocysts_NaFAc RAW_FILE_NAME=PP_invitrocysts_NaFAc_1 SUBJECT_SAMPLE_FACTORS - PP_invitrocysts_NaFAc_2 Factor:PP_invitrocysts_NaFAc RAW_FILE_NAME=PP_invitrocysts_NaFAc_2 SUBJECT_SAMPLE_FACTORS - PP_invitrocysts_NaFAc_3 Factor:PP_invitrocysts_NaFAc RAW_FILE_NAME=PP_invitrocysts_NaFAc_3 SUBJECT_SAMPLE_FACTORS - PP_invitrocysts_NaFAc_4 Factor:PP_invitrocysts_NaFAc RAW_FILE_NAME=PP_invitrocysts_NaFAc_4 SUBJECT_SAMPLE_FACTORS - PP_tachys_NaFAc_1 Factor:PP_tachys_NaFAc RAW_FILE_NAME=PP_tachys_NaFAc_1 SUBJECT_SAMPLE_FACTORS - PP_tachys_NaFAc_2 Factor:PP_tachys_NaFAc RAW_FILE_NAME=PP_tachys_NaFAc_2 SUBJECT_SAMPLE_FACTORS - PP_tachys_NaFAc_3 Factor:PP_tachys_NaFAc RAW_FILE_NAME=PP_tachys_NaFAc_3 SUBJECT_SAMPLE_FACTORS - PP_uninf_NaFAc_1 Factor:PP_uninf_NaFAc RAW_FILE_NAME=PP_uninf_NaFAc_1 SUBJECT_SAMPLE_FACTORS - PP_uninf_NaFAc_2 Factor:PP_uninf_NaFAc RAW_FILE_NAME=PP_uninf_NaFAc_2 SUBJECT_SAMPLE_FACTORS - NP_invitrocysts_mock_1 Factor:NP_invitrocysts_mock RAW_FILE_NAME=NP_invitrocysts_mock_1 SUBJECT_SAMPLE_FACTORS - NP_invitrocysts_mock_2 Factor:NP_invitrocysts_mock RAW_FILE_NAME=NP_invitrocysts_mock_2 SUBJECT_SAMPLE_FACTORS - NP_invitrocysts_mock_3 Factor:NP_invitrocysts_mock RAW_FILE_NAME=NP_invitrocysts_mock_3 SUBJECT_SAMPLE_FACTORS - NP_invitrocysts_mock_4 Factor:NP_invitrocysts_mock RAW_FILE_NAME=NP_invitrocysts_mock_4 SUBJECT_SAMPLE_FACTORS - NP_tachys_mock_1 Factor:NP_tachys_mock RAW_FILE_NAME=NP_tachys_mock_1 SUBJECT_SAMPLE_FACTORS - NP_tachys_mock_2 Factor:NP_tachys_mock RAW_FILE_NAME=NP_tachys_mock_2 SUBJECT_SAMPLE_FACTORS - NP_tachys_mock_3 Factor:NP_tachys_mock RAW_FILE_NAME=NP_tachys_mock_3 SUBJECT_SAMPLE_FACTORS - NP_tachys_mock_4 Factor:NP_tachys_mock RAW_FILE_NAME=NP_tachys_mock_4 SUBJECT_SAMPLE_FACTORS - NP_tachys_mock_5 Factor:NP_tachys_mock_5 RAW_FILE_NAME=NP_tachys_mock_5 SUBJECT_SAMPLE_FACTORS - NP_uninf_mock_1 Factor:NP_uninf_mock RAW_FILE_NAME=NP_uninf_mock_1 SUBJECT_SAMPLE_FACTORS - NP_uninf_mock_2 Factor:NP_uninf_mock RAW_FILE_NAME=NP_uninf_mock_2 SUBJECT_SAMPLE_FACTORS - NP_Blank_1 Factor:NP_Blank RAW_FILE_NAME=NP_Blank_1 SUBJECT_SAMPLE_FACTORS - NP_Blank_2 Factor:NP_Blank RAW_FILE_NAME=NP_Blank_2 SUBJECT_SAMPLE_FACTORS - NP_Blank_3 Factor:NP_Blank RAW_FILE_NAME=NP_Blank_3 SUBJECT_SAMPLE_FACTORS - NP_invitrocysts_NaFAc_1 Factor:NP_invitrocysts_NaFAc RAW_FILE_NAME=NP_invitrocysts_NaFAc_1 SUBJECT_SAMPLE_FACTORS - NP_invitrocysts_NaFAc_2 Factor:NP_invitrocysts_NaFAc RAW_FILE_NAME=NP_invitrocysts_NaFAc_2 SUBJECT_SAMPLE_FACTORS - NP_invitrocysts_NaFAc_3 Factor:NP_invitrocysts_NaFAc RAW_FILE_NAME=NP_invitrocysts_NaFAc_3 SUBJECT_SAMPLE_FACTORS - NP_invitrocysts_NaFAc_4 Factor:NP_invitrocysts_NaFAc RAW_FILE_NAME=NP_invitrocysts_NaFAc_4 SUBJECT_SAMPLE_FACTORS - NP_tachys_NaFAc_1 Factor:NP_tachys_NaFAc RAW_FILE_NAME=NP_tachys_NaFAc_1 SUBJECT_SAMPLE_FACTORS - NP_tachys_NaFAc_2 Factor:NP_tachys_NaFAc RAW_FILE_NAME=NP_tachys_NaFAc_2 SUBJECT_SAMPLE_FACTORS - NP_tachys_NaFAc_3 Factor:NP_tachys_NaFAc RAW_FILE_NAME=NP_tachys_NaFAc_3 SUBJECT_SAMPLE_FACTORS - NP_uninf_NaFAc_1 Factor:NP_uninf_NaFAc RAW_FILE_NAME=NP_uninf_NaFAc_1 SUBJECT_SAMPLE_FACTORS - NP_uninf_NaFAc_2 Factor:NP_uninf_NaFAc RAW_FILE_NAME=NP_uninf_NaFAc_2 #COLLECTION CO:COLLECTION_SUMMARY For metabolome measurements of tissue cysts and tachyzoites, tachyzoite CO:COLLECTION_SUMMARY isolation, cyst maturation and isolation were performed as described above. CO:COLLECTION_SUMMARY Metabolites were extracted in 80 % acetonitrile (Carl Roth) and 20 % water (Carl CO:COLLECTION_SUMMARY Roth) containing internal standards (phenolphthalein, CAPS, PIPES CO:COLLECTION_SUMMARY (Sigma-Aldrich)). Cell pellets were sonicated for 5 min and after centrifugation CO:COLLECTION_SUMMARY (21,500 x g, 5 min, 0 °C), the supernatants were transferred to MS vials for CO:COLLECTION_SUMMARY immediate LC/MS analysis. 5 µl of each sample were collected to generate a CO:COLLECTION_SUMMARY pooled biological quality control (PBQC). 20 µl of the in vitro cysts, bead CO:COLLECTION_SUMMARY control and host cell background samples, and 5 µl of the tachyzoite samples CO:COLLECTION_SUMMARY were injected. The injection order of the samples was randomized, blanks and CO:COLLECTION_SUMMARY PBQCs were injected periodically. The samples were analyzed on a Q-Exactive Plus CO:COLLECTION_SUMMARY mass spectrometer (Thermo Fisher Scientific) via 70k MS1 scans, with CO:COLLECTION_SUMMARY intermittent 35k data-dependent 35k MS2 scans in positive and negative mode CO:COLLECTION_SUMMARY separately. Chromatographic separation was achieved on a Vanquish Flex fitted CO:COLLECTION_SUMMARY with an ACQUITY UPLC BEH Amide column (Waters). Running a 25 min linear gradient CO:COLLECTION_SUMMARY starting with 90 % eluent A (10 mM ammonium carbonate in acetonitrile) / 10 % CO:COLLECTION_SUMMARY eluent B (10 mM ammonium carbonate in water) and ending with 40 % eluent A / 60 CO:COLLECTION_SUMMARY % eluent B, followed by washing and equilibration steps. XCalibur 4.2.47 CO:COLLECTION_SUMMARY software and Compound Discoverer 3.1 software (Thermo Fisher Scientific) was CO:COLLECTION_SUMMARY used for recording and used for peak detection, combination of adducts and CO:COLLECTION_SUMMARY compound annotation, respectively. Metabolite identifications were based on CO:COLLECTION_SUMMARY either retention time and accurate mass match to an in-house library of 160 CO:COLLECTION_SUMMARY authentic standards, or by matching accurate mass and MS2 fragments to m/z cloud CO:COLLECTION_SUMMARY database (mirrored offline in m/z vault v 2.1.22.15 in May 2020) (Thermo Fisher CO:COLLECTION_SUMMARY Scientific). CO:SAMPLE_TYPE Toxoplasma gondii #TREATMENT TR:TREATMENT_SUMMARY Cells were either treated or untreated tachyzoites or treated or untreated TR:TREATMENT_SUMMARY bradyzoites (cysts) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Uninfected and infected myotubes were prepared. In both cases, two T150 dishes SP:SAMPLEPREP_SUMMARY were pooled into one sample. For bradyzoite samples, myotubes were infected with SP:SAMPLEPREP_SUMMARY 3.2*106 Pru-tdTomato tachyzoites corresponding to a MOI of 0.3 and cyst SP:SAMPLEPREP_SUMMARY formation was induced for indicated time. On the day of harvest, infected SP:SAMPLEPREP_SUMMARY samples and uninfected host cell controls were placed on ice, medium was removed SP:SAMPLEPREP_SUMMARY and monolayers were washed three times with ice-cold PBS. Cells were then SP:SAMPLEPREP_SUMMARY harvested by scraping into 10 ml ice-cold 0.05 % BSA in PBS per T150 dish. Cysts SP:SAMPLEPREP_SUMMARY were released from the monolayer via forcing through a 23G needle (Sterican®) SP:SAMPLEPREP_SUMMARY 25 times with a syringe and collected via centrifugation (1,200 x g, 10 min, 0 SP:SAMPLEPREP_SUMMARY °C). The supernatant was removed, the pellet was resuspended carefully in SP:SAMPLEPREP_SUMMARY ice-cold 2 % BSA in PBS containing 200 µl DBA-coupled beads (preparation SP:SAMPLEPREP_SUMMARY described below) and samples were incubated for 1 h at 4 °C with gentle SP:SAMPLEPREP_SUMMARY shaking. Subsequently, the samples were placed in a magnetic stand on ice, SP:SAMPLEPREP_SUMMARY washed five times with 0.1 % BSA in PBS to remove cell debris, followed by two SP:SAMPLEPREP_SUMMARY washing steps with PBS to remove residual BSA. Cysts and beads were then SP:SAMPLEPREP_SUMMARY collected via centrifugation (1,200 x g, 10 min, 0 °C), shock frozen in liquid SP:SAMPLEPREP_SUMMARY nitrogen and stored at -80 °C until extraction. Tachyzoite samples were SP:SAMPLEPREP_SUMMARY generated in T150 dishes by infecting myotubes and HFF cells with 3.2*107 SP:SAMPLEPREP_SUMMARY tachyzoites corresponding to an MOI of 3 for 48 h. Medium was replaced by SP:SAMPLEPREP_SUMMARY ice-cold PBS and monolayers were scraped and passaged through a 27G needle. SP:SAMPLEPREP_SUMMARY Tachyzoites were filter-purified through a 3 µm filter (Whatman) and PBS-washed SP:SAMPLEPREP_SUMMARY by centrifugation (1,200 x g, 10 min, 0 °C) three times. All samples were SP:SAMPLEPREP_SUMMARY extracted simultaneously in 80 % acetonitrile for LC/MS analysis as described SP:SAMPLEPREP_SUMMARY below. Bead-only controls were processed equally. Bead-supplemented tachyzoite SP:SAMPLEPREP_SUMMARY controls were processed equally to cyst samples, replacing washing steps via SP:SAMPLEPREP_SUMMARY magnetic stand by centrifugation (1,200 x g, 10 min, 0 °C). Preparation of SP:SAMPLEPREP_SUMMARY beads Coupling of DynabeadsTM MyONETM Streptavdin T1 (Thermo Fisher Scientific) SP:SAMPLEPREP_SUMMARY to DBA was done as described in the manufacturer's protocol. Briefly, 200 µl SP:SAMPLEPREP_SUMMARY beads/sample were resuspended in 1 ml PBS by vortexing, washed three times with SP:SAMPLEPREP_SUMMARY PBS in a magnetic stand and resuspended in 1 ml PBS containing 50 µg DBA / SP:SAMPLEPREP_SUMMARY sample. The tube containing the DBA-magnetic bead mixture was incubated on a SP:SAMPLEPREP_SUMMARY rotary mixer for 45 min at RT. Uncoupled DBA was removed by washing the coated SP:SAMPLEPREP_SUMMARY beads three times with PBS. After washing, the DBA-coated beads were resuspended SP:SAMPLEPREP_SUMMARY in 2 ml PBS containing 2 % BSA. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation was achieved on a Vanquish Flex fitted with an CH:CHROMATOGRAPHY_SUMMARY ACQUITY UPLC BEH Amide column (Waters). Running a 25 min linear gradient CH:CHROMATOGRAPHY_SUMMARY starting with 90 % eluent A (10 mM ammonium carbonate in acetonitrile) / 10 % CH:CHROMATOGRAPHY_SUMMARY eluent B (10 mM ammonium carbonate in water) and ending with 40 % eluent A / 60 CH:CHROMATOGRAPHY_SUMMARY % eluent B, followed by washing and equilibration steps. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS The injection order of the samples was randomized, blanks and PBQCs were MS:MS_COMMENTS injected periodically. The samples were analyzed on a Q-Exactive Plus mass MS:MS_COMMENTS spectrometer (Thermo Fisher Scientific) via 70k MS1 scans, with intermittent 35k MS:MS_COMMENTS data-dependent 35k MS2 scans in positive and negative mode separately.XCalibur MS:MS_COMMENTS 4.2.47 software and Compound Discoverer 3.1 software (Thermo Fisher Scientific) MS:MS_COMMENTS was used for recording and used for peak detection, combination of adducts and MS:MS_COMMENTS compound annotation, respectively. Metabolite identifications were based on MS:MS_COMMENTS either retention time and accurate mass match to an in-house library of 160 MS:MS_COMMENTS authentic standards, or by matching accurate mass and MS2 fragments to m/z cloud MS:MS_COMMENTS database (mirrored offline in m/z vault v 2.1.22.15 in May 2020) (Thermo Fisher MS:MS_COMMENTS Scientific). Data were exported to Excel for grouping, combination of datasets MS:MS_COMMENTS from positive and negative ionization runs and blank subtraction.. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS counts MS_METABOLITE_DATA_START Samples PP_invitrocysts_mock_1 PP_invitrocysts_mock_2 PP_invitrocysts_mock_3 PP_invitrocysts_mock_4 PP_tachys_mock_1 PP_tachys_mock_2 PP_tachys_mock_3 PP_tachys_mock_4 PP_tachys_mock_5 PP_uninf_mock_1 PP_uninf_mock_2 PP_Blank_1 PP_Blank_2 PP_Blank_3 PP_invitrocysts_NaFAc_1 PP_invitrocysts_NaFAc_2 PP_invitrocysts_NaFAc_3 PP_invitrocysts_NaFAc_4 PP_tachys_NaFAc_1 PP_tachys_NaFAc_2 PP_tachys_NaFAc_3 PP_uninf_NaFAc_1 PP_uninf_NaFAc_2 NP_invitrocysts_mock_1 NP_invitrocysts_mock_2 NP_invitrocysts_mock_3 NP_invitrocysts_mock_4 NP_tachys_mock_1 NP_tachys_mock_2 NP_tachys_mock_3 NP_tachys_mock_4 NP_tachys_mock_5 NP_uninf_mock_1 NP_uninf_mock_2 NP_Blank_1 NP_Blank_2 NP_Blank_3 NP_invitrocysts_NaFAc_1 NP_invitrocysts_NaFAc_2 NP_invitrocysts_NaFAc_3 NP_invitrocysts_NaFAc_4 NP_tachys_NaFAc_1 NP_tachys_NaFAc_2 NP_tachys_NaFAc_3 NP_uninf_NaFAc_1 NP_uninf_NaFAc_2 Factors Factor:PP_invitrocysts_mock Factor:PP_invitrocysts_mock Factor:PP_invitrocysts_mock Factor:PP_invitrocysts_mock Factor:PP_tachys_mock Factor:PP_tachys_mock Factor:PP_tachys_mock Factor:PP_tachys_mock Factor:PP_tachys_mock_5 Factor:PP_uninf_mock Factor:PP_uninf_mock Factor:PP_Blank Factor:PP_Blank Factor:PP_Blank Factor:PP_invitrocysts_NaFAc Factor:PP_invitrocysts_NaFAc Factor:PP_invitrocysts_NaFAc Factor:PP_invitrocysts_NaFAc Factor:PP_tachys_NaFAc Factor:PP_tachys_NaFAc Factor:PP_tachys_NaFAc Factor:PP_uninf_NaFAc Factor:PP_uninf_NaFAc Factor:NP_invitrocysts_mock Factor:NP_invitrocysts_mock Factor:NP_invitrocysts_mock Factor:NP_invitrocysts_mock Factor:NP_tachys_mock Factor:NP_tachys_mock Factor:NP_tachys_mock Factor:NP_tachys_mock Factor:NP_tachys_mock_5 Factor:NP_uninf_mock Factor:NP_uninf_mock Factor:NP_Blank Factor:NP_Blank Factor:NP_Blank Factor:NP_invitrocysts_NaFAc Factor:NP_invitrocysts_NaFAc Factor:NP_invitrocysts_NaFAc Factor:NP_invitrocysts_NaFAc Factor:NP_tachys_NaFAc Factor:NP_tachys_NaFAc Factor:NP_tachys_NaFAc Factor:NP_uninf_NaFAc Factor:NP_uninf_NaFAc Glycine 5134818 4320930 5465900 2788015 3791259 13792232 11797835 12281033 Crotonic acid 1275701333 1298599912 323861061 501048492 590741161 510841092 Pyruvic acid 13908602 5373828 8328907 6492501 10243256 142163334 107514777 129103314 121505405 46517353 81278225 3481212 4229088 4158542 5413190 20509624 214663784 276737836 279113928 20468000 31310112 Alanine 16730304 6719160 13018143 544419409 35745427 508243658 29748041 137037632 1783376 32910224 21118532 26191986 12360069 90443169 76112923 41798813 29222063 36078345 22232199 29282950 47571729 41641505 44544132 Lactic acid 4079588908 4292527445 5475430890 3609469104 3020486238 5159187410 3990177102 4148764257 GABA 17445223 35445166 16072323 14413765 119716445 96553914 14131540 31090461 31757009 7942992 37642045 13887447 29258163 14944358 13084587 16452298 36411228 30511811 15118913 11486385 3701880 78557694 92018604 85358164 72443576 105044826 1760769 129409522 118316956 125933878 Choline 1317185108 879912940 1040578568 99689716 1465041058 1487320329 104651189 410340686 90848399 117088030 1688335094 1798814791 1616347031 1314237168 1612078613 25784878 78795818 Serine 2415302 691216 1123779 7344930 870494 2463886 5380611 2034763 2973728 2812918 6162017 5324671 6268869 Diethanolamine 7839707357 1621564938 1279115458 1413214381 6673944796 4978776685 2790242220 1635489178 215602978 1422018 1336638996 1977231282 1619277804 1461230270 3266716511 3378373775 174303432 165555428 Cytosine 67188160 22757366 10955728 187506115 13329428 124816145 4160136 106366283 12803504 44318095 6615270 6823961 Histamine 14961404 7832717 139301780 110985684 150815248 51504996 2692705 12732343 2813721 82916201 Uracil 28405694 3542487 26569223 17871460 992623 1752444 2023761 Creatinine 11181076 16182207 66983306 69192216 59924069 19146713 17768815 2823681 11124296 2632758 5806915 490408 3354045 3788430 4021825 3339230 3310306 5771376 5010147 5768823 Proline 285814042 195445192 466922398 1201575238 631184918 700233601 483117944 518858429 66354004 180386665 413673670 345554399 446489938 243916842 1046472498 1048503447 1199268070 4835906 1609945 5913171 10088773 7100636 7484579 4805177 7051114 4049331 3085240 8275011 5154021 14581303 16264759 14207253 Fumaric acid 46327308 15075305 59877363 161325878 170290368 185833410 150675049 173926753 30876592 52064808 33714989 27823332 39330433 190451271 243342892 208509700 Hexanoic acid 136374026 135480539 178795398 139110332 25608957 47650909 30428571 28661187 25564022 67022757 85590342 21339267 6190755 91363316 114979930 130783891 119415922 39812098 34803647 39232418 91342215 74520044 Valine 165813923 193593690 575440044 89216195 661208870 431145731 537793698 234900641 438593541 43397134 62541397 658252975 636650630 747043569 224025783 526030543 967372292 967442145 6731953 Betaine 204777828 75963663 119907834 93426559 303514606 215636402 348342871 175149464 358454175 3319175 218552721 14780186 9882213 120336746 99014704 66458396 203688289 408979401 315569597 420830206 121782178 Succinic acid 119284168 84715260 2643687937 3143032215 2889836773 2716412950 3513451321 158927854 273340604 4746896 26643232 390002083 1852055026 1346442888 1856241129 Threonine 11647683 9288855 12295838 5064707 9175995 45683413 33122468 36977503 Nicotinamide 124269987 68288903 82554390 1178846489 410584727 991045736 912766252 1036647222 176995837 181133125 112323970 110710339 405257683 403375081 78131115 51255638 89189547 80420789 67949047 70822239 31093978 67714408 Nicotinic acid 44249069 3778294 18971221 574062 15410891 2695110 8453140 5852641 1165451 1790999 1148996 140631190 53423857 78451283 45078908 61131815 1322537 1184220 739743 1104457 48741888 61722754 49212538 Taurine 144107445 137070901 164869031 28266110 148151691 12271441 169919149 16548804 122058247 5568902 20256490 213219561 179628205 193333047 143671629 334551906 336265822 246073653 3766945 793928 168550195 146022103 213352049 34887721 134673481 149526604 144898716 132459247 138776590 9179904 19464320 178337343 193615357 245257638 209134819 529438072 472299721 484084235 10493467 14266899 Methylcytosine 32701976 30697920 26891245 26920443 24351721 Oxoproline 1777448 165693562 127417942 93919568 1226423 1603584 447806 2908139 6693913 6743172078 7335237506 6682861447 5703538424 6434407792 772144 931836 1380488 7384576551 5786002935 6767399043 650580 Acetylputrescine 705909479 573052331 1064041409 107888374 31512708 3865964 29511020 5993701 29918785 13191583 36035360 39425727 1611116 1271461239 1396894438 1147199770 874133377 43243998 6036173 23462982 10764368 Acetylalanine 117846038 54314826 62812751 57695005 73740033 47264355 43260025 41514134 Creatine 286758425 226302593 385911167 27745921 621136015 602866324 322988953 454335128 24440604 50598825 223212317 504069458 458733887 380322241 2608153618 2292849023 19616767 20015359 2955636 2009210 4099356 3200614 3436280 3229020 2984853 3535368 3663020 3783328 4419646 3636962 25445333 23405396 24656025 Isoleucine 214839674 124166292 445627101 1067015297 655683485 948427739 602778028 729931660 26064983 629551080 639595021 532586301 304936419 2107920705 1592429501 2046075394 Leucine 121529105 567337842 360375803 575687125 43198990 538019840 704726880 1593886528 1315063191 Aspartic acid 927077585 609375630 1051804635 456992024 757126189 1406427532 1204560888 1283316743 Hypoxanthine 81084512 705647 55852139 4995046 114015620 155977788 569266 94708815 379715249 18615361 47877928 34702035 23834074 40907526 97644839 17378560 771365 11930945 19258017 17917276 15156022 15143244 4262758 2715237 187595912 166439380 182767299 Methylnicotinamide 444054098 367457710 557965340 1600423729 910014883 1576051723 531030159 1121360271 1403520 1048844 728877301 722576865 773325068 614862485 5002367498 4249610998 4864989368 Hydroxypicolinic acid 125310042 175336623 190984901 205383377 2770136 2063480 1367916 2467926 1870550 289377356 273626585 3158816 4728079 4764016 162510792 158914111 138711933 158107944 4507493 3906854 5125567 387507323 208899107 Caprylic acid 41660195814 29477201869 53729986983 47959302152 5159787 8711101 11722023 1916932442 13809781934 8823994 79324403394 58584765655 25147592464 47173899206 12658936 92592600 999734036 5801366969 aKG 17337348 5464874 1207096742 758747809 1343142255 943457215 1178113328 44007823 7770938 52243848 8032041 794567053 817064977 1068716187 Glutamine 15725248 6520673 45632107 6292383 8389222 7533246 4801849 7919686 28038053 17125659 46918211 39516512 81972519 69795548 71034825 Lysine 2177835 483562738 183940870 6485385781 3817093 40811406 8964240 11678761 1715387671 8274822795 2141467 Glutamic acid 26236071 79210609 8182936358 6080391974 8272524006 5163078349 6629189992 75813445 65460124 89835592 53914212 10751441696 7626830177 7653233635 29818936 2999563 36130874 4023649371 3729525715 4164209299 2911768842 4057556746 39258930 23909993 69146485 43371978 4935533179 4689596214 5036002562 Orotic acid 3564606 2275215 2604089 1250172 23356820 17704139 10625803 Nonanoic acid 1728648121 1416943617 3288254174 1652237531 578754515 793143726 726507096 692730488 822558397 2056477205 1961320267 275841687 298551658 321032384 1890324095 1583315258 1594410511 2806093678 888255046 791387072 961620617 3198692272 1746998543 Methylglutamine 825457 49937758 36686563 2263725 32280405 1152244 1844616 5130940 3942554 1576752 Carnitine 186067919 103414443 196710667 2207851 724200808 473627341 892935830 378173454 606072728 12405618 22659249 98593174 238768170 227807399 204936286 1120290295 1026186620 1073913667 11752468 4826856 Phenylalanine 120872049 73787078 20613288 11169574 824575516 147742679 100757135 94175673 636764763 11668359 25669091 234778165 220951147 255848771 109686031 582976031 832151551 430186520 2588279 23472076 62146198 3481220 130081889 118540413 137140108 94478114 117115236 4718318 8559007 43421629 93695773 52054170 223144904 233257164 194665899 1715478 Glycerol 3 phosphate 23031806 11241797 31784458 1452886 25037547 24762809 26149222 22426441 25963465 568963 534319 23920070 20544893 29624040 25966798 43980690 38306166 42639511 791345 Decanoic acid 6654892 2362951 1983772 5607702 2779658 1745086 961335 2079058 3028027 9001925 8378308 6160970 6741055 1841451 2123399 2447730 3425750 496058060 415952691 564349754 473970638 150292316 230362723 201488555 198477144 188231823 449299387 478509261 26748351 26623265 26779193 594613190 458734814 413698718 670832722 230910707 205222168 224740506 712500586 429811985 Aconitic acid 175696 118632 127830 356134 19097925 16311872 18104576 12542697 15661133 606537 600573 629096 649718 92882299 105432314 103728184 Arginine 60089784 29654939 66108685 535546280 264919651 479149406 252370694 288447821 121039484 49635240 65596819 53078189 399807141 386223970 273170158 20560395 19406838 19283072 14619892 13581393 38828091 25098252 28348222 Acetylaspartic acid 10726899 900886 7585808 5874888 13135293 18618056 11671255 18526026 11233033 23146249 183134723 169705635 187413951 21532831 18263787 22590759 23809692 735150198 671021138 690767064 Acetylglutamine 240067 369584 93225439 109149653 65639717 418974 350375 385731 2609245 15793711 30616427 33233987 33216919 25837276 28083052 34568258 31883697 36060056 Trimethyllysine 10170832 1970512803 958849657 2535450667 667372672 824132614 792978668 469741294 Threonine 3 phosphate 10253842 7334875 9942052 600331 3836885 1962107 4621759 2266226 2299344 13294887 11737832 11963017 10388001 2905815 2663523 11191262 7320777 12344623 870484 3923731 3726031 4966803 3728585 3499498 667110 10703326 10836494 13269690 13097098 3940724 3265285 3734566 678122 Lauric acid 375142981 404959191 295942162 422347827 129271077 138550711 121548970 333011567 233212195 549043630 280863620 12600441 11776446 12385050 462772752 509750384 370353943 347438752 256793544 281188796 287560528 623997597 354765986 Dimethylarginine 194322390 64542714 165542270 50263756 108212931 76171154 84345537 38999174 Acetylcarnitine 34127224 28100292 52908250 2935897 438663462 293098736 488448748 217779734 321349803 2656494 186665112 252950996 258938712 201483580 299762128 288559574 Glycerophosphorylethanolamine 3667284 1593689 5756453 33334564 13988211 35582805 9458037 24008820 10166896 4887817 3132918 5169399 31181037 29681697 7558438 1887256 11382016 20472331 19938189 19585131 16891036 21780433 9693480 5398284 12046384 9679688 37202297 31310934 35041917 Acetylarginine 448239 596139 800880 73856169 19423577 78326143 14991894 48439050 1064246 808493 449836 1023862 27708138 31347029 1360422 1289754 1525460 1144424 1466432 1371252 1223880 1269675 Pantothenic acid 273966062 297188853 218160276 125092441 110398919 589059067 602621698 693201091 522197529 639864352 725039998 637811815 683743880 Deoxyuridine 4312275 41739293 46121118 32598893 27630405 43150237 5816627 4323954 2306636 52462206 39856966 57329028 Myristic acid 464937078 511343308 631738546 514022866 417925635 486092050 442975138 386125463 476647202 611804871 509773899 526240927 772722478 531308507 409198403 758138082 660889798 740546389 728775947 458610254 Cytidine 38698357 36446978 84715958 4050321 212534061 52288395 148351296 11399018 87604566 94278081 50534463 85469207 147816337 100611641 51362774 14858054 28609946 47140337 37721826 39269275 18049133 29985739 24961291 24141238 43232462 29305919 46380248 59922317 50392439 Uridine 4036583 12790736 5880080 499253 5154861 4618754 2388208 131713 733844 2076064 12544684 3119877 332780243 209030335 196620101 117714916 171569257 13009876 379254362 335590692 349285971 Glycerophosphoglycerol 160745980 85767730 158580015 6681559 2107433 4063476 1321578 1489275 1627793 159414160 96151991 148334580 120731454 2333268 2037169 2383295 1248796 1756471 Palmitic acid 8320143832 9936500317 12934624401 12932818363 5117821287 4857784636 4650511850 3592821483 4788986750 6885781121 7294844578 16064491 9737861033 21001409031 11327559515 7764014675 4537073063 4871512679 4991423211 6082896931 6637563651 Adenosine 426255020 2056426251 994404589 2082962987 230790086 1784789571 18020642 332279346 491840434 121502001 2998342154 3517991055 4486401 17792019 300230976 371091312 287891228 186198169 300806847 47818339 2199704 43761171 858202385 813008709 868714287 Inosine 182199683 130068275 173742006 219877114 3941700 265923158 4966207 186719767 9074076 13871835 236628989 395941801 284014448 112040515 544943915 481431704 434226086 1590872 679237 125272658 56750895 89750127 204967139 193744435 204863084 178740749 192677025 264816718 313731508 315265142 340958124 920250307 705566825 768932636 6683437 Linoleic acid 1751390061 1930982191 2942994249 758887020 238211975 253670888 257667316 257728369 246682574 738442390 553796792 3149559225 2679934351 3837153100 2748870305 325764336 290644913 318741987 831045356 Oleic acid 2600330335 2831187592 4321980321 1554069731 4698713622 4990621435 5186320565 4742967070 5070990366 1946564766 1397296262 5188616 5652740887 4285272250 5898724254 5580777163 5031930696 4371775886 4945371712 1986947135 1588568053 Guanosine 10398761 8753607 34394454 1333987 68397524 25005784 56115880 13393848 33631745 32804164 42299494 34408266 21663815 98577947 101876790 98286159 19751807 11753412 21696473 1170176 28997032 25837966 24210720 12933676 24025691 15802712 23387616 36183996 23977634 72046206 69554141 82476672 Stearic acid 5233472960 6176801056 7009139337 10170074156 2160237321 2031122423 1757401105 1250727786 1677091063 4042688901 5222896800 5625535 94012935 94978702 6689640309 16633584406 7480881520 6055895612 1674575417 2036881955 1839987747 3383744706 4113922091 Tretinoin 515896331 327791464 737830881 245324900 294624557 433386293 251065705 230505897 120264682 276741794 6682196 6375179 6311758 142040269 442833444 519380173 238477841 191037189 228152668 246308213 N-Acetylglucosamine 1 phosphate 16496357 6523913 16374173 3324661 2776905 2930708 2200266 3720635 16271275 14102361 16420989 14359985 17340362 11919106 14590806 Eicosapentaenoic acid 988085039 1061510950 1722019523 271556049 98049137 104090992 99453390 91731230 98545621 253435488 1710884 1745878798 1339690135 1877884081 1973783708 152065113 134185406 143483186 424923845 357359210 Arachidonic acid 1698280180 1893964495 2525677377 397038385 394632633 450740620 426308918 381323913 414395283 323999543 1975966 2104638 1964467 2550753045 2549358431 3860761226 2590407155 510016419 435940794 458656081 419673529 Arachidic acid 45889492 70821933 80400104 113075482 35638972 32504666 33476415 26912663 34182900 36902686 47371092 1652623 1602349 78164109 133051625 73831268 82089588 17406545 20388076 20235860 37647932 34155227 Uridine monophosphate (UMP) 13662304 7602983 8039666 9264612 13716501 9163876 cAMP 3742342 5919789 6303711 5259604 5456825 4468601 3385951 4085083 Adenosine 5'-monophosphate 79407052 27146903 39041470 22019125 40830243 23132803 3603311 7113224 Nervonic acid 19069902 28371436 31782154 106351524 104225078 122512399 102639090 112979417 37507440 31992919 34868871 45871616 74958033 70530846 67583233 3759544 UDPGlucNAc 13446661 4313249 13035920 2444583 7175693 14119930 12002574 12643931 1643615 3288154 37208731 22318329 26039726 Coenzyme Q7 3581293 2861982 30645265 3434797 4376292 2457722 2137767 2628872 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention time (min) MW Glycine 15.0 75.0319 Crotonic acid 15.5 86.0371 Pyruvic acid 1.7 88.0161 Alanine 14.4 89.0478 Lactic acid 4.2 90.0318 GABA 15.3 103.0636 Choline 19.3 103.1000 Serine 15.1 105.0427 Diethanolamine 15.4 105.0788 Cytosine 8.2 111.0436 Histamine 20.9 111.0802 Uracil 4.2 112.0276 Creatinine 6.7 113.0593 Proline 13.1 115.0635 Fumaric acid 8.4 116.0111 Hexanoic acid 1.9 116.0838 Valine 12.6 117.0791 Betaine 11.6 117.0792 Succinic acid 6.8 118.0267 Threonine 14.0 119.0584 Nicotinamide 10.9 122.0480 Nicotinic acid 4.5 123.0320 Taurine 9.8 125.0144 Methylcytosine 15.4 125.0591 Oxoproline 5.2 129.0426 Acetylputrescine 19.5 130.1106 Acetylalanine 4.1 131.0583 Creatine 14.6 131.0694 Isoleucine 11.1 131.0946 Leucine 10.6 131.0946 Aspartic acid 14.5 133.0375 Hypoxanthine 7.0 136.0384 Methylnicotinamide 20.5 136.0635 Hydroxypicolinic acid 1.8 139.0270 Caprylic acid 1.8 144.1149 aKG 7.8 146.0216 Glutamine 15.1 146.0692 Lysine 15.4 146.1056 Glutamic acid 13.6 147.0530 Orotic acid 4.1 156.0171 Nonanoic acid 1.7 158.1306 Methylglutamine 7.3 160.0845 Carnitine 15.0 161.1051 Phenylalanine 10.3 165.0788 Glycerol 3 phosphate 15.9 172.0137 Decanoic acid 1.6 172.1463 Aconitic acid 8.0 174.016438 Arginine 24.4 174.1117 Acetylaspartic acid 10.2 175.0476 Acetylglutamine 5.9 188.0797 Trimethyllysine 24.9 188.1524 Threonine 3 phosphate 14.5 199.0245 Lauric acid 1.6 200.1776 Dimethylarginine 23.0 202.1439 Acetylcarnitine 13.0 203.1158 Glycerophosphorylethanolamine 15.5 215.0558 Acetylarginine 15.5 216.1223 Pantothenic acid 4.5 219.1105 Deoxyuridine 11.4 228.0746 Myristic acid 1.6 228.2088 Cytidine 9.7 243.0853 Uridine 6.4 244.0693 Glycerophosphoglycerol 8.1 246.0502 Palmitic acid 1.6 256.2400 Adenosine 7.1 267.0960 Inosine 8.8 268.0804 Linoleic acid 1.6 280.2399 Oleic acid 1.5 282.2555 Guanosine 11.0 283.0915 Stearic acid 1.5 284.2711 Tretinoin 1.6 300.2086 N-Acetylglucosamine 1 phosphate 15.5 301.0560 Eicosapentaenoic acid 1.5 302.2242 Arachidonic acid 1.5 304.2399 Arachidic acid 1.5 312.3024 Uridine monophosphate (UMP) 15.4 324.0356 cAMP 5.0 329.0524 Adenosine 5'-monophosphate 15.1 347.0629 Nervonic acid 1.5 366.3494 UDPGlucNAc 13.7 607.0816 Coenzyme Q7 2.0 658.4957 METABOLITES_END #END