#METABOLOMICS WORKBENCH wilk0211_20190227_114504 DATATRACK_ID:1654 STUDY_ID:ST001148 ANALYSIS_ID:AN001895 PROJECT_ID:PR000767 VERSION 1 CREATED_ON March 12, 2019, 1:01 pm #PROJECT PR:PROJECT_TITLE Effect of harvesting technique and storage on the metabolic profile of PR:PROJECT_TITLE patient-derived skin fibroblasts PR:PROJECT_TYPE MS targeted metabolomics PR:PROJECT_SUMMARY Using human skin fibroblasts, we measured the effect of different cell PR:PROJECT_SUMMARY harvesting techniques (trypsinization, scraping, and methanol fixation) as well PR:PROJECT_SUMMARY as storage time on the metabolic profiles of organic acids, amino acids, and PR:PROJECT_SUMMARY acylcarnitines. PR:INSTITUTE Mayo Clinic - Rochester PR:DEPARTMENT Neurology PR:LAST_NAME Wilkins PR:FIRST_NAME Jordan PR:ADDRESS 200 First St SW PR:EMAIL wilkins.jordan@mayo.edu PR:PHONE 5072933857 #STUDY ST:STUDY_TITLE Effect of cell harvesting technique and storage on metabolic profiles in human ST:STUDY_TITLE skin fibroblasts ST:STUDY_TYPE Method ST:STUDY_SUMMARY Human skin fibroblasts were cultured in MEM media supplemented with 15% FBS. ST:STUDY_SUMMARY Cells were harvested using (i) trypsinization, (ii) scraping, (iii) methanol ST:STUDY_SUMMARY fixation and scraping, (iV) methanol fixation, scraping, and drying. Targeted ST:STUDY_SUMMARY metabolomics analysis of organic acids, amino acids, and acycarnitines was ST:STUDY_SUMMARY conducted at Mayo Clinic Metabolomics Core Facility. ST:INSTITUTE Mayo Clinic ST:DEPARTMENT Neurology ST:LAST_NAME Wilkins ST:FIRST_NAME Jordan ST:ADDRESS 200 First St SW ST:EMAIL wilkins.jordan@mayo.edu ST:PHONE 5072933857 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 21 SU:GENDER Male SU:CELL_BIOSOURCE_OR_SUPPLIER Mayo Clinic SU:CELL_STRAIN_DETAILS 10268 SU:CELL_PASSAGE_NUMBER 10 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 10268_1 Harvest_technique:Trypsin | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_2 Harvest_technique:Trypsin | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_3 Harvest_technique:Trypsin | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_4 Harvest_technique:Scraping | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_5 Harvest_technique:Scraping | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_6 Harvest_technique:Scraping | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_7 Harvest_technique:Methanol | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_8 Harvest_technique:Methanol | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_9 Harvest_technique:Methanol | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_10 Harvest_technique:Methanol_dry | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_11 Harvest_technique:Methanol_dry | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_12 Harvest_technique:Methanol_dry | Storage_time:48_hours SUBJECT_SAMPLE_FACTORS - 10268_13 Harvest_technique:Trypsin | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_14 Harvest_technique:Trypsin | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_15 Harvest_technique:Trypsin | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_16 Harvest_technique:Scraping | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_17 Harvest_technique:Scraping | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_18 Harvest_technique:Scraping | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_19 Harvest_technique:Methanol | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_20 Harvest_technique:Methanol | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_21 Harvest_technique:Methanol | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_22 Harvest_technique:Methanol_dry | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_23 Harvest_technique:Methanol_dry | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_24 Harvest_technique:Methanol_dry | Storage_time:2_weeks SUBJECT_SAMPLE_FACTORS - 10268_25 Harvest_technique:Trypsin | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_26 Harvest_technique:Trypsin | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_27 Harvest_technique:Trypsin | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_28 Harvest_technique:Scraping | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_29 Harvest_technique:Scraping | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_30 Harvest_technique:Scraping | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_31 Harvest_technique:Methanol | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_32 Harvest_technique:Methanol | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_33 Harvest_technique:Methanol | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_34 Harvest_technique:Methanol_dry | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_35 Harvest_technique:Methanol_dry | Storage_time:4_weeks SUBJECT_SAMPLE_FACTORS - 10268_36 Harvest_technique:Methanol_dry | Storage_time:4_weeks #COLLECTION CO:COLLECTION_SUMMARY Prior to harvest, media was removed and cells were rinsed with PBS. For CO:COLLECTION_SUMMARY trypsinization, cells were dissociated by incubation with 0.05% trypsin at 37ºC CO:COLLECTION_SUMMARY for 5 minutes. Dissociated cells were resuspended in MEM (containing 15% FBS) to CO:COLLECTION_SUMMARY neutralize trypsin activity. Cells were centrifuged at 1000 x g for 5 min at CO:COLLECTION_SUMMARY 4ºC. Pelleted cells were resuspended in PBS and centrifuged at 1000 x g for 5 CO:COLLECTION_SUMMARY min at 4°C. The supernatant was aspirated, cell pellets were flash frozen in CO:COLLECTION_SUMMARY liquid nitrogen, and frozen pellets were stored at -80ºC. For scraping, cells CO:COLLECTION_SUMMARY were detached in 1 mL PBS using a rubber scraper. Detached cells were CO:COLLECTION_SUMMARY centrifuged at 1000 x g for 5 min at 4°C, and PBS was aspirated. Cell pellets CO:COLLECTION_SUMMARY were flash frozen in liquid nitrogen and stored at -80ºC. For methanol CO:COLLECTION_SUMMARY fixation, 1 mL 80% methanol (pre-chilled on dry ice) was added to cells. Tissue CO:COLLECTION_SUMMARY culture plates were rocked by hand for about 20 seconds to evenly distribute CO:COLLECTION_SUMMARY methanol solution. Cells were detached in 80% methanol using a rubber scraper. CO:COLLECTION_SUMMARY Suspensions were transferred to an Eppendorf tube and stored at -80ºC. For CO:COLLECTION_SUMMARY methanol fixation and drying, cells were harvested in 1 mL 80% methanol, and the CO:COLLECTION_SUMMARY supernatant was evaporated using a Speed Vacuum at room temperature. Dried CO:COLLECTION_SUMMARY samples were stored at -80ºC. Frozen samples were stored at -80ºC for 48 CO:COLLECTION_SUMMARY hours, two weeks, or four weeks. CO:SAMPLE_TYPE Cultured fibroblasts CO:COLLECTION_FREQUENCY 48 hours, 2 weeks, and 4 weeks CO:STORAGE_CONDITIONS -80℃ CO:STORAGE_VIALS 1.5 mL Eppendorf tubes #TREATMENT TR:TREATMENT_SUMMARY Collection method and storage time were variable as described in collection TR:TREATMENT_SUMMARY summary #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Targeted metabolomics analysis of amino acids, acylcarnitines, and metabolites SP:SAMPLEPREP_SUMMARY of the TCA cycle was conducted at the Mayo Clinic Metabolomics Center. Samples SP:SAMPLEPREP_SUMMARY harvested in methanol were dried using a Speed Vacuum. Cell pellets were SP:SAMPLEPREP_SUMMARY sonicated in 100 µL PBS and spiked with 15 - 25 µL of the respective internal SP:SAMPLEPREP_SUMMARY standards. Proteins were removed by adding 450 µL of cold 1:1 SP:SAMPLEPREP_SUMMARY methanol/acetonitrile solution with subsequent centrifugation for 15 minutes SP:SAMPLEPREP_SUMMARY (18,000 x g at 4ºC). Supernatants were transferred to a 1 mL dram and dried SP:SAMPLEPREP_SUMMARY under a nitrogen stream for approximately 30 minutes. Prior to detection, TCA SP:SAMPLEPREP_SUMMARY cycle metabolites were derivatized using ethoxyamine and then with MtBSTFA + 1% SP:SAMPLEPREP_SUMMARY tBDMCS (N-Methyl-N-(t-Butyldimethylsilyl)-Trifluoroacetamide + 1% SP:SAMPLEPREP_SUMMARY t-Butyldimethylchlorosilane). Amino acids were derivatized with SP:SAMPLEPREP_SUMMARY 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using the Waters AccQ-Fluor SP:SAMPLEPREP_SUMMARY Reagent Kit (Cat. # WATO52880). Acylcarnitines were reconstituted in buffer SP:SAMPLEPREP_SUMMARY containing 99% MeOH, 1% H2O, 1 mM ammonium formate, and 0.1% formic acid. SP:SAMPLEPREP_SUMMARY Organic acids were detected with an Agilent 5977A gas chromatography/mass SP:SAMPLEPREP_SUMMARY spectrometry (GC/MS) under electron impact and single ion monitoring conditions. SP:SAMPLEPREP_SUMMARY Analytes were separated on an Agilent DB-5MS column (30 m x 0.25 mm x 0.25μm). SP:SAMPLEPREP_SUMMARY Sample injection volume was 1 μL performed in splitless mode. The inlet SP:SAMPLEPREP_SUMMARY temperature was maintained at 250°C. The carrier gas was helium set at a flow SP:SAMPLEPREP_SUMMARY rate of 0.9 ml/min. The initial oven temperature was 120°C set with the SP:SAMPLEPREP_SUMMARY following ramp rates: Ramp to 180°C at 25°C/min; Ramp to 270°C at 6°C/min; SP:SAMPLEPREP_SUMMARY Ramp to 325°C at 30°C/min. The transfer line temperature was 280°C. SP:SAMPLEPREP_SUMMARY Concentrations of lactic acid, fumaric acid, succinic acid, oxaloacetic acid, SP:SAMPLEPREP_SUMMARY alpha-ketoglutaric acid, malic acid, 2-hydroxyglutaric acid, cis-aconitic acid, SP:SAMPLEPREP_SUMMARY citric acid, and isocitric acid were measured against a calibration curve. Amino SP:SAMPLEPREP_SUMMARY acids were analyzed using Thermo TSQ Quantum Ultra mass spectrometer (West Palm SP:SAMPLEPREP_SUMMARY Beach, FL) coupled with a Waters ACQUITY ultra performance liquid chromatography SP:SAMPLEPREP_SUMMARY BEH C18 column (2.1 mm x 150 mm x. 1.7 μm). Data acquisition was performed SP:SAMPLEPREP_SUMMARY using selected reaction monitoring (SRM) and positive electrospray ionization SP:SAMPLEPREP_SUMMARY (ESI). Injection volume was 1 μL. The column flow rate was 400 μL/min with an SP:SAMPLEPREP_SUMMARY isothermal set at 43°C. Mobile phase A was 1% acetonitrile in 0.1% formic acid, SP:SAMPLEPREP_SUMMARY and mobile phase B was 100% acetonitrile. The mass spectrometer was operated SP:SAMPLEPREP_SUMMARY with 4000 capillary voltage, 50 sheath gas, 20 auxiliary gas, and 15 sweep gas. SP:SAMPLEPREP_SUMMARY The capillary temperature was 2700°C. Collision gas was 1.5 Torr and collision SP:SAMPLEPREP_SUMMARY energy was 25 V. The tube lens was kept at 90 V. The concentration of amino SP:SAMPLEPREP_SUMMARY acids was calculated against a calibration curve. Acylcarnitines were analyzed SP:SAMPLEPREP_SUMMARY using a Waters ACQUITY UPLC system (Milford, MA) coupled with a Thermo TSQ SP:SAMPLEPREP_SUMMARY Quantiva tandem mass spectrometer (West Palm Beach, FL) in SRM and positive SP:SAMPLEPREP_SUMMARY (H)ESI mode. Analytes were separated on a Waters ACQUITY UPLC BEH C8 column (2.1 SP:SAMPLEPREP_SUMMARY mm x 150 mm x. 1.7 μm) with an isothermal temperature of 43°C. The mass SP:SAMPLEPREP_SUMMARY spectrometer capillary voltage was set to 4000 with a sheath gas 30, auxiliary SP:SAMPLEPREP_SUMMARY gas 5, and sweep gas 2. The ion transfer tube was maintained at 300°C with the SP:SAMPLEPREP_SUMMARY vaporizer at 40°C, collision gas at 1.5 Torr, and collision energy at 12 V. SP:SAMPLEPREP_SUMMARY Concentrations of carnitine, acetylcarnitine, propionylcarnitine, SP:SAMPLEPREP_SUMMARY butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, lauroylcarnitine, SP:SAMPLEPREP_SUMMARY myristoylcarnitine, palmitoylcarnitine, oleoylcarnitine, and stearoylcarnitine SP:SAMPLEPREP_SUMMARY were measured against a calibration curve. Data sets collected by GC/MS were SP:SAMPLEPREP_SUMMARY analyzed using Mass Hunter GC/MS Quantitation software version B.07 (Agilent). SP:SAMPLEPREP_SUMMARY Analysis of LC/MS data sets was performed using Xcalibur Quant browser (Thermo SP:SAMPLEPREP_SUMMARY Scientific). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Acylcarnitines were analyzed using a Waters ACQUITY UPLC system (Milford, MA) CH:CHROMATOGRAPHY_SUMMARY coupled with a Thermo TSQ Quantiva tandem mass spectrometer (West Palm Beach, CH:CHROMATOGRAPHY_SUMMARY FL) in SRM and positive (H)ESI mode. Analytes were separated on a Waters ACQUITY CH:CHROMATOGRAPHY_SUMMARY UPLC BEH C8 column (2.1 mm x 150 mm x. 1.7 μm) with an isothermal temperature CH:CHROMATOGRAPHY_SUMMARY of 43°C. The mass spectrometer capillary voltage was set to 4000 with a sheath CH:CHROMATOGRAPHY_SUMMARY gas 30, auxiliary gas 5, and sweep gas 2. The ion transfer tube was maintained CH:CHROMATOGRAPHY_SUMMARY at 300°C with the vaporizer at 40°C, collision gas at 1.5 Torr, and collision CH:CHROMATOGRAPHY_SUMMARY energy at 12 V. Concentrations of carnitine, acetylcarnitine, CH:CHROMATOGRAPHY_SUMMARY propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, CH:CHROMATOGRAPHY_SUMMARY lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, oleoylcarnitine, and CH:CHROMATOGRAPHY_SUMMARY stearoylcarnitine were measured against a calibration curve. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C8 (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Quantum Ultra MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Xcalibur acquisition and Quantitation 2.0.7 (Thermo Electron) #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS micromol metabolite per gram protein MS_METABOLITE_DATA_START Samples 10268_1 10268_2 10268_3 10268_4 10268_5 10268_6 10268_7 10268_8 10268_9 10268_10 10268_11 10268_12 10268_13 10268_14 10268_15 10268_16 10268_17 10268_18 10268_19 10268_20 10268_21 10268_22 10268_23 10268_24 10268_25 10268_26 10268_27 10268_28 10268_29 10268_30 10268_31 10268_32 10268_33 10268_34 10268_35 10268_36 Factors Harvest_technique:Trypsin | Storage_time:48_hours Harvest_technique:Trypsin | Storage_time:48_hours Harvest_technique:Trypsin | Storage_time:48_hours Harvest_technique:Scraping | Storage_time:48_hours Harvest_technique:Scraping | Storage_time:48_hours Harvest_technique:Scraping | Storage_time:48_hours Harvest_technique:Methanol | Storage_time:48_hours Harvest_technique:Methanol | Storage_time:48_hours Harvest_technique:Methanol | Storage_time:48_hours Harvest_technique:Methanol_dry | Storage_time:48_hours Harvest_technique:Methanol_dry | Storage_time:48_hours Harvest_technique:Methanol_dry | Storage_time:48_hours Harvest_technique:Trypsin | Storage_time:2_weeks Harvest_technique:Trypsin | Storage_time:2_weeks Harvest_technique:Trypsin | Storage_time:2_weeks Harvest_technique:Scraping | Storage_time:2_weeks Harvest_technique:Scraping | Storage_time:2_weeks Harvest_technique:Scraping | Storage_time:2_weeks Harvest_technique:Methanol | Storage_time:2_weeks Harvest_technique:Methanol | Storage_time:2_weeks Harvest_technique:Methanol | Storage_time:2_weeks Harvest_technique:Methanol_dry | Storage_time:2_weeks Harvest_technique:Methanol_dry | Storage_time:2_weeks Harvest_technique:Methanol_dry | Storage_time:2_weeks Harvest_technique:Trypsin | Storage_time:4_weeks Harvest_technique:Trypsin | Storage_time:4_weeks Harvest_technique:Trypsin | Storage_time:4_weeks Harvest_technique:Scraping | Storage_time:4_weeks Harvest_technique:Scraping | Storage_time:4_weeks Harvest_technique:Scraping | Storage_time:4_weeks Harvest_technique:Methanol | Storage_time:4_weeks Harvest_technique:Methanol | Storage_time:4_weeks Harvest_technique:Methanol | Storage_time:4_weeks Harvest_technique:Methanol_dry | Storage_time:4_weeks Harvest_technique:Methanol_dry | Storage_time:4_weeks Harvest_technique:Methanol_dry | Storage_time:4_weeks 3_Methylhistidine 0.533779394 0.439071548 0.511756276 0.382503699 0.758822025 0.587269265 0.508721234 0.538014811 0.603169938 0.422325015 0.372939769 0.467888463 0.523010167 0.184502609 0.208932167 0.449327626 0.608118962 0.618891902 0.72129855 0.564494558 0.462714326 0.546470568 0.436477443 0.509622487 0.131323006 0.115024839 0.121237811 0.824867742 0.769172802 0.61475803 0.524700447 0.804127322 0.59368129 5_Hydroxylysine 0.086327258 0.064702016 0.074532084 0.057532427 0.067077867 0.054425151 0.040706657 0.05260844 0.043661975 0.050094904 0.040190414 0.043659746 0.084465284 0.071608476 0.076595161 0.063524257 0.062935449 0.058846732 0.079048124 0.056302408 0.046868112 0.06294594 0.060179334 0.065008056 0.085561347 0.070327531 0.079873861 0.052905152 0.046794253 0.04474456 0.066655355 0.042883704 0.060625331 0.063919546 0.051330866 0.056244626 Alanine 31.47807353 25.74883057 27.55331865 8.387843642 6.871570147 6.623138009 54.2073098 59.9013055 48.84962334 49.15435393 49.08038868 56.92376484 35.82474395 33.13899152 34.69720764 18.65529369 17.35815926 16.91103115 47.1851455 43.2444813 54.06522368 50.96072592 54.10764231 52.53827809 30.30732171 31.47882813 34.01417262 11.66547466 10.15128275 11.83772986 48.9823994 44.84308289 42.80911548 47.43600019 48.50443773 45.62039255 alpha_Aminobutyric_acid 0.371049461 0.314142079 0.341117681 0.316485833 0.269622487 0.246763867 0.414113015 0.482833929 0.401456664 0.40066255 0.438981657 0.511028111 0.566618304 0.401603648 0.437971777 0.134177885 0.158284119 0.119307099 0.580388689 0.57441956 0.585120653 0.631763047 0.54260631 0.580186555 0.352663854 0.348217131 0.347697465 0.172650813 0.16978413 0.174376339 0.415960375 0.347225631 0.373972888 0.412781996 0.406863612 0.410783246 Aminoadipic_acid 1.314082771 1.19837238 1.266987993 0.504320117 0.408881305 0.369819082 0.991560758 1.074756959 0.905373789 0.889644349 0.844244469 0.973852785 1.622730703 1.613336842 1.634211418 0.266902907 0.312104358 0.349389924 1.143433433 1.066141233 1.310007059 1.212451009 1.212419589 1.260995097 1.247625331 1.202095544 1.375621809 0.318694435 0.334121024 0.331755374 1.081235142 1.144623008 1.015982578 0.984204017 1.198073712 1.07285566 Arginine 13.02185287 10.42047602 10.58372567 6.25169491 5.680182485 5.588898793 25.99218697 25.40720047 21.81496982 22.97942315 24.04209371 23.86431363 16.68447824 14.01734623 14.60109728 21.01953569 19.93647756 20.69828784 17.23387053 18.61133869 19.33658214 19.65348154 26.81179373 19.98717158 14.47512072 13.39003794 14.90922761 15.77549222 12.24629256 14.80568292 21.43965816 16.86022898 19.68191512 19.67943375 22.89220465 18.17550818 Asparagine 16.61375656 15.68079604 15.47296539 2.544721257 2.94951473 2.773641268 19.89244198 21.34233394 17.96067844 18.65237781 18.54461449 21.75905682 22.81462213 22.00273896 17.84653631 5.681366148 4.105916147 5.464484093 18.40751477 17.01411017 19.67501833 16.09142112 20.72192028 18.48831016 17.64054363 17.12264915 18.92161073 5.443519565 4.967593551 5.011701892 19.03697155 16.49045549 16.79267211 17.89035814 17.99732604 17.49510848 Aspartic_Acid 35.73670763 29.37827729 31.54905857 3.672238154 3.17701276 3.055123165 18.28250132 20.17993131 15.37152115 15.06429849 14.01304585 18.25780175 37.06814693 33.32486139 30.40731583 3.754631797 3.895848386 4.162131905 14.96711377 14.5174921 16.5057627 16.0508403 16.79205347 16.58515634 34.05149611 32.8542823 35.55856531 3.822140075 4.04787592 4.504295539 15.81381721 15.51629729 14.78246367 14.98505658 15.39531871 16.09629737 beta_Alanine 1.819419725 1.423533046 1.379525481 1.386256256 1.419568177 1.266544423 1.260970243 1.154466748 1.563354119 2.050123723 1.665750815 1.513576064 1.356203542 1.241519622 1.456555081 1.337695084 1.533310755 1.381250588 1.327047215 1.341659422 1.292954911 1.149175399 1.011842222 0.929818226 1.166559817 0.903531574 1.193822846 beta_Aminoisobutyric_acid 0.004318769 0.001592988 0.002760519 0.006034444 0.009657759 0.007997947 0.007280387 0.008977298 0.009810988 0.000785424 0.001072586 0.005113481 0.004699833 0.009995043 0.009279365 0.003477337 0.003108155 0.002520779 0.004120978 0.004545548 0.003500751 Carnosine 0.197208577 0.145649183 0.180912086 0.386863014 0.297299405 0.285009426 0.187115386 0.234836215 0.180684222 0.198815957 0.202478971 0.188254479 0.198304145 0.122957266 0.121780263 0.133418586 0.123354146 0.139260607 0.169334205 0.160097419 0.168483249 0.159155111 0.152997415 0.130200529 0.095917774 0.113378113 0.072239521 0.072944578 0.071658322 0.138702362 0.114272263 0.116112692 0.126261891 0.138077937 0.134550396 Citrulline 0.438655452 0.268754362 0.306812679 0.095282395 0.084811179 0.073224091 0.665349082 0.695300118 0.498735289 0.482188685 0.593488522 0.557478159 0.412318994 0.359768523 0.284077708 0.027359446 0.018114583 0.002518612 0.420366287 0.44684559 0.576899603 0.46069667 0.52395545 0.492878416 0.345936389 0.306166458 0.381659612 0.009924577 0.001448443 0.010786166 0.569001441 0.488614896 0.441364661 0.530467484 0.513491485 0.521236411 Cystathionine 1.64041351 1.343783428 1.644112662 0.770010139 0.90365468 0.796974076 1.011870141 0.611243285 1.027446124 1.721859361 1.5714604 1.437705917 0.245738916 0.273023469 0.284861496 0.80259853 0.619973511 0.6580397 0.736108537 0.719288108 0.778992118 1.662787489 1.405852898 1.430763727 0.222539665 0.179712119 0.157578641 0.815107643 0.677535625 0.712452829 0.777522676 0.584262383 0.826899872 Cysteine 0.025026397 0.367259607 0.469714333 0.312991431 0.320946314 0.656669722 0.198076847 0.19744767 0.040584142 0.245049725 0.204544041 0.424795388 0.338136573 0.467441697 0.225184107 0.37909394 0.559600739 0.481838608 0.192525268 0.186371564 0.283166057 0.256011114 0.149488844 0.305958737 0.41620729 0.352557457 0.338869291 0.341639803 0.508602044 0.322474624 Ethanolamine 0.269813334 0.206664027 0.250098686 0.237397564 0.201830812 0.176829759 0.147471232 0.153392994 0.109505012 0.107879653 0.124533833 0.118354671 0.346027828 0.240175237 0.283824326 0.107574849 0.120360363 0.131549861 0.204691094 0.12240886 0.08421474 0.084582311 0.110550855 0.095538797 0.22500802 0.225128401 0.230180769 0.130900973 0.136777269 0.165635866 0.137527769 0.083400334 0.118828591 0.102114267 0.120651541 0.085016694 gamma_Aminobutyric_acid 0.810347214 0.602833801 0.721833584 0.238664589 0.187822139 0.185079181 0.591481961 0.661953192 0.54878097 0.496935452 0.475383111 0.58393239 0.966193745 0.86907615 0.952939918 0.598095972 0.587652735 0.721784329 0.624312508 0.665339075 0.679448276 0.710233858 0.721338256 0.846482737 0.146915945 0.161315326 0.177479307 0.593271444 0.564298006 0.584760479 0.545837283 0.587323782 0.641768541 Glutamic_Acid 429.3647733 345.9381579 373.0740717 33.89739849 30.18535885 29.99468255 328.4397465 331.4762628 277.4287141 271.0570427 254.6406507 324.5389266 405.8426979 363.295583 346.2780961 48.32679957 47.96183583 50.68732874 240.3172496 230.0953931 282.7235816 234.0983062 274.8645701 250.2628979 346.5838322 333.7887098 374.6886869 36.57678602 39.78464628 44.56140698 289.6008982 267.5453255 257.0282006 269.8445126 270.2467095 286.926194 Glutamine 183.3770388 172.2145197 170.4542046 6.293540218 10.95111461 8.797240622 283.7998903 311.8766451 240.5385467 276.7369314 220.9493126 260.5150295 262.1206953 224.265512 207.6347197 282.7834395 273.2094094 277.7334218 267.4765117 314.2765941 322.1026885 211.6662253 216.9280014 228.7830562 19.81431887 18.47317155 19.48464254 316.9819178 282.4974039 264.5915766 313.7868397 298.2007418 294.8289276 Glycine 71.47232555 59.63577183 62.02603354 8.871492946 8.490098057 6.934050316 67.71501139 76.57361299 64.13170314 64.79951338 54.09078628 64.29146579 76.59241529 68.27237635 67.14140939 19.24502513 16.17773741 18.86138754 59.78570423 63.51641515 69.6152355 67.4489102 70.90757923 63.56438737 62.98638801 62.25192716 68.18069879 14.2565867 12.92818096 14.30507546 66.51574501 63.90989487 59.63053772 63.59911469 62.48856882 65.40471386 Histidine 12.68409843 10.38412536 10.71984782 2.531127426 2.199871503 2.197328144 20.01501944 20.17108706 18.04490596 17.20535755 16.84291654 16.68698394 12.24204975 11.55934879 10.11833687 4.536270363 4.084787756 4.22784251 14.27540648 13.53873515 14.607085 12.17233239 16.01032575 12.94480966 10.33635365 10.19112778 13.4989958 3.071432866 2.728243315 3.209889248 15.63658368 15.13064661 13.93534275 12.89321618 17.2503777 13.88691742 Hydroxyproline 3.153248523 2.521121629 2.372264707 0.161853076 0.159835467 0.199572495 3.022424904 3.631744306 3.12742128 2.379927396 2.693871343 3.221771405 3.689625572 3.545301332 3.050028125 2.601042816 3.516535099 3.754251681 2.957655027 3.228185399 3.665881082 2.843890099 3.1504413 3.172995483 0.033035908 3.463518413 3.344972603 2.816332629 3.074854811 3.277983806 3.034187486 Isoleucine 17.50363586 14.49755313 15.90731466 3.046215618 3.089224672 2.859229445 20.58412116 23.7859717 19.26383604 18.8953086 20.18943598 20.09994325 20.53261511 19.18830731 19.06826256 6.201807036 5.64083498 6.46051914 18.08554099 17.70666513 18.79630865 19.47815926 21.2873164 19.24146811 15.91320309 15.8747736 17.3719457 4.802872823 3.90049557 4.700932569 19.03327244 16.63590427 16.46230355 18.00041553 18.71662289 17.23703394 Leucine 20.02830465 16.43658045 18.00918502 7.080061767 6.610559896 5.758803616 21.66009489 25.28842935 20.11944049 21.14235425 22.2926308 21.79950386 23.98327261 23.81504575 21.79587304 17.689507 16.39790427 17.91531801 18.65772841 19.1705188 20.4377021 20.8746117 22.64295889 20.07609867 19.17133228 18.79288824 20.59225365 11.75573091 9.214025986 11.0507317 20.21460627 18.16942347 17.5992231 19.05840584 19.80505981 18.49011257 Lysine 8.155118681 7.571395098 7.850952191 6.150048483 5.824729988 5.748464401 16.16156494 19.26185524 14.66260949 14.92894438 17.39582414 16.8368891 13.02007728 10.97448909 11.89339717 12.93176504 12.33013393 12.35124516 14.86807084 14.34552475 14.98108881 15.52277722 19.37427128 15.78734001 9.806239774 9.987178074 11.07286222 10.18192889 8.92506516 9.055757369 17.86291687 12.60941586 13.78832885 15.6374959 17.06906851 13.94349356 Methionine 4.670398423 3.777183957 4.255487607 1.749701552 1.463712187 1.369550635 6.533073398 7.999873682 5.881193089 5.831197544 6.183837836 7.151218505 6.637946668 6.031870448 5.671457857 5.115134338 5.210512395 5.507130052 5.750384907 5.174004277 5.976612511 6.308877908 6.070807891 5.981496215 5.892331809 5.543946529 5.793240566 3.987765888 3.356798837 3.785027666 6.436741259 5.814287325 5.743077478 6.228310092 5.854831416 5.892361192 Ornithine 2.371908662 1.908763247 2.097505516 0.549513538 0.605613296 0.6794491 4.340160636 4.553531317 3.502690095 3.798810652 4.262553241 4.546581858 3.16244299 2.960549847 2.710191727 0.690048629 0.607083509 0.783313179 3.990380369 3.66359177 3.986353934 4.116237665 4.465877097 3.915234719 2.166066726 2.17694126 2.2162808 0.569756338 0.462854806 0.541816447 3.691340099 3.185277551 3.451096025 3.854661445 3.98554272 3.367189451 Phenylalanine 10.68383001 9.220240106 9.660919546 3.342990761 2.816088772 2.715001804 14.11664113 15.61893333 13.14033834 13.57865668 13.27346616 14.15116078 13.32028334 12.50164126 12.45549112 7.272178098 6.804425483 6.946748767 12.77285895 12.98581853 14.23671286 14.36186993 15.53702547 14.25893907 11.17035992 11.16914618 12.46631872 5.448434891 4.631029279 5.238667371 14.88193413 12.90149711 12.49562979 13.48339926 13.89867815 13.04524019 Phosphoethanolamine 6.054379014 6.966472284 7.086250949 1.07499668 1.300686997 1.15739088 1.191530051 1.302963958 1.379693494 1.921342997 0.885226151 1.599960581 8.957040852 5.289315084 8.462225494 1.270849889 1.749521584 1.322815462 3.108458679 2.418971733 2.29403148 2.639454717 2.63553485 2.271100566 6.443941756 5.762489272 4.331087463 1.034212313 1.758666502 1.695366048 1.838966683 1.827414863 1.606819155 1.657598847 1.502816853 1.616231104 Proline 50.93097316 44.15027443 47.48928421 5.111990747 4.514500623 4.187000503 44.64944772 46.27185327 42.29575751 38.35147758 35.08309834 43.0566288 51.86749308 50.28892367 49.45930124 10.16107606 9.884602575 10.10637141 37.538342 37.85562675 45.25185811 38.71721403 40.03746456 39.59365496 43.08023051 44.17941714 45.92747961 7.340131667 6.89905421 7.684117922 40.73180651 36.76541041 35.28797434 35.76001796 38.3531692 37.27228413 Sarcosine 0.116192195 0.094140484 0.097305313 0.123885287 0.14614489 0.113731834 0.107783873 0.094928483 0.119621375 0.130766894 0.11952564 0.111377661 0.024284165 0.024103077 0.022497753 0.104430932 0.104375916 0.122699881 0.115318407 0.11128806 0.109503736 0.092578797 0.092025472 0.104789245 7.71E-05 0.001243837 0.002330533 0.112761078 0.102501586 0.102075301 0.097601764 0.098424595 0.103709479 Serine 12.94327531 10.16525394 9.865400095 4.245149771 3.834346806 3.321964486 11.48450804 13.38387953 10.23425351 11.59403497 9.779579311 11.75641838 17.75407373 17.33044058 15.74260126 30.06689643 28.45319038 33.10583976 9.354139169 10.35995579 10.71033067 10.5961659 10.19657466 10.99032442 16.16405386 14.91667414 15.66587897 17.91525633 14.54257643 17.12341475 10.43039912 8.882163079 9.017720976 9.626780658 10.28830532 9.757046529 Taurine 22.47077323 16.63210195 17.6776638 0.516302142 0.757835238 0.559410541 23.8810112 24.57121878 19.85613078 19.11725169 18.3685599 20.50559536 18.91912946 19.70851537 16.191696 2.191592899 2.085492659 1.895338161 16.78569746 20.49840641 21.87645377 20.37997427 21.17549813 19.25744761 17.50125205 18.34163325 20.91504049 0.436855386 0.492340145 0.932703379 23.14925414 25.81368577 21.74089908 21.84776041 25.67723107 22.44540391 Threonine 36.21367572 29.73790819 31.37656493 9.310571102 8.214782973 8.520459399 44.83573286 50.91247165 41.19600556 42.40802737 39.46875122 46.11064144 44.46441206 38.77700475 38.40967129 17.86226863 17.24940091 18.59676599 41.03881477 37.65516474 44.46897482 43.01000645 45.855349 43.04653955 35.9682768 36.52105667 38.76151722 13.50749793 12.13238308 14.32687452 43.75814405 39.54357211 39.00518222 41.94628079 42.4515611 42.59171464 Tryptophan 3.384881445 2.945745795 3.133971447 0.093771492 0.154454168 0.181292647 4.580042576 4.881940518 3.998223472 3.973774307 4.440776809 4.248198462 4.099151885 3.477557129 3.279513791 0.876381416 0.833100168 0.828475962 3.677452418 3.861399913 4.140672117 4.114674991 4.5037649 4.177738815 3.233346846 3.382939811 3.727412957 0.67290195 0.625075461 0.78902562 4.314459119 3.958781145 3.747357891 4.095222274 4.23630836 3.858953722 Tyrosine 12.17143483 10.4841817 11.02783368 3.181994096 2.882516378 2.292884524 14.41426664 18.34513671 14.49425843 15.08623536 13.86531801 15.29705059 15.15647086 13.77796054 12.69963503 9.891863088 9.57663147 10.29243857 12.51407497 13.0047742 13.91098966 14.00360955 14.70561616 15.34099757 12.88043405 12.41846808 13.50711263 7.465059919 5.641464143 6.625452265 14.91721662 12.89889566 12.54459488 13.36916463 13.53959467 13.15281269 Valine 14.70127486 13.033024 14.28211117 4.738429969 4.449935665 3.871609168 19.20853412 23.52113489 19.43183025 17.2599933 19.96502752 22.67878494 21.14308078 17.58528738 18.0903438 13.66515245 12.19628359 12.103815 19.66408563 18.47904407 19.40953923 20.89167912 20.63043968 19.46570826 16.09714842 16.47707146 17.87197401 9.041143328 6.624877214 8.155636735 18.46883542 16.87226695 16.63965165 18.30227576 19.24351194 17.23149137 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name rtimes (mins) Transition HMDB 3_Methylhistidine 4.59 340>171 HMDB00479 5_Hydroxylysine 16.46 252>171 HMDB00450 Alanine 13.71 260>171 HMDB00161 alpha_Aminobutyric_acid 17.49 274>171 HMDB00452 Aminoadipic_acid 15.33 332>171 HMDB00510 Arginine 5.38 345>171 HMDB00517 Asparagine 4.75 303>171 HMDB00168 Aspartic_Acid 8.35 304>171 HMDB00191 beta_Alanine 10.67 260>171 HMDB00056 beta_Aminoisobutyric_acid 15.6 274>171 HMDB03911 Carnosine 5.44 199>171 HMDB00033 Citrulline 9.92 346>171 HMDB00904 Cystathionine 17.68 282>171 HMDB00099 Cysteine 18.91 291>171 HMDB00574 Ethanolamine 6.68 232>171 HMDB00149 gamma_Aminobutyric_acid 14.27 274>171 HMDB00112 Glutamic_Acid 10.2 318>171 HMDB00148 Glutamine 6.15 317>171 HMDB00641 Glycine 6.79 246>171 HMDB00123 Histidine 3.9 164>171 HMDB00177 Hydroxyproline 4.12 302>171 HMDB00725 Isoleucine 25.32 302>171 HMDB00172 Leucine 26.02 302>171 HMDB00687 Lysine 18.87 244>171 HMDB00182 Methionine 20.2 320>171 HMDB00696 Ornithine 17.58 237>171 HMDB00214 Phenylalanine 27.52 335>171 HMDB00159 Phosphoethanolamine 4.91 312>171 HMDB00224 Proline 15.65 286>171 HMDB00162 Sarcosine 9.07 260>171 HMDB00271 Serine 6.06 276>171 HMDB00187 Taurine 5.7 296>171 HMDB00251 Threonine 12.08 290>171 HMDB00167 Tryptophan 29.18 375>171 HMDB00929 Tyrosine 19.9 352>171 HMDB00158 Valine 20.64 288>171 HMDB00883 METABOLITES_END #END