#METABOLOMICS WORKBENCH Sethjparker_20241101_121629 DATATRACK_ID:5330 STUDY_ID:ST003548 ANALYSIS_ID:AN005832 PROJECT_ID:PR002184 VERSION 1 CREATED_ON November 5, 2024, 3:03 pm #PROJECT PR:PROJECT_TITLE Restricting lysine normalizes toxic catabolites associated with PR:PROJECT_TITLE ALDH7A1-deficiency in cells and mice PR:PROJECT_TYPE Manuscript PR:PROJECT_SUMMARY Lysine metabolism converges at α-aminoadipic semialdehyde dehydrogenase PR:PROJECT_SUMMARY (ALDH7A1). Rare loss-of-function mutations in ALDH7A1 cause a toxic accumulation PR:PROJECT_SUMMARY of lysine catabolites, including piperideine 6-carboxylate (P6C), that are PR:PROJECT_SUMMARY thought to cause fatal seizures in children unless strictly managed with dietary PR:PROJECT_SUMMARY lysine reduction. In this study, we perform metabolomics and expression analysis PR:PROJECT_SUMMARY of tissues from Aldh7a1-deficient mice, which reveal tissue-specific differences PR:PROJECT_SUMMARY in lysine metabolism and other metabolic pathways. We also develop a fluorescent PR:PROJECT_SUMMARY biosensor to characterize lysine transporter activity and identify competitive PR:PROJECT_SUMMARY substrates that reduce the accumulation of lysine catabolites in PR:PROJECT_SUMMARY ALDH7A1-deficient HEK293 cells. Lastly, we show that intravenous administration PR:PROJECT_SUMMARY of lysine α-oxidase from Trichoderma viride reduces lysine and P6C levels by PR:PROJECT_SUMMARY >80% in mice. Our results improve our understanding of lysine metabolism and PR:PROJECT_SUMMARY make inroads towards improving therapeutic strategies for lysine catabolic PR:PROJECT_SUMMARY disorders. PR:INSTITUTE University of British Columbia PR:DEPARTMENT Biochemistry & Molecular Biology PR:LABORATORY Parker laboratory PR:LAST_NAME Parker PR:FIRST_NAME Seth PR:ADDRESS 950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada PR:EMAIL seth.parker@bcchr.ca PR:PHONE 6048753121 #STUDY ST:STUDY_TITLE Metabolomics of Aldh7a1-/- mice treated with lysine alpha-oxidase from ST:STUDY_TITLE Trichoderma viride ST:STUDY_SUMMARY Metabolite analysis of brain, liver, and plasma isolated from Aldh7a1-/- ST:STUDY_SUMMARY (n=3/each) fed diets consisting of 0.9% w/w lysine and 18 ppm pyridoxine ST:STUDY_SUMMARY administered with PBS or 150 U/kg lysine alpha-oxidase (LOX) from Trichoderma ST:STUDY_SUMMARY viride. Tissues were cryo-homogenized using a liquid nitrogen cooled mortar and ST:STUDY_SUMMARY pestle into a fine powder and approximately 25-50 mg of each tissue was ST:STUDY_SUMMARY transferred to a pre-weighed homogenization tube to which ~20 2.3 mm zirconia ST:STUDY_SUMMARY beads and 1 mL of 80% ice cold methanol were added. Tissues were homogenized ST:STUDY_SUMMARY using a bead mill and cleared by centrifugation at 4C, where each of the 3-4 ST:STUDY_SUMMARY cycles consisted of a 60 second homogenization at 6 m/s followed by a 30 second ST:STUDY_SUMMARY pause. A volume equivalent to 25 mg of tissue extract was transferred to a new ST:STUDY_SUMMARY tube, and 250 ul of 80% methanol containing 1 ul of stable isotope-labeled ST:STUDY_SUMMARY internal amino acid standard mix was added. Metabolites were concentrated using ST:STUDY_SUMMARY a SpeedVac until dry. For plasma metabolite analysis, 10 µl of plasma was added ST:STUDY_SUMMARY to 250 µl of 80% methanol containing 1 µl of stable isotope-labeled internal ST:STUDY_SUMMARY amino acid standard mix, vortexed at 4C for 10 minutes, and centrifuged. 0.9 ml ST:STUDY_SUMMARY of supernatant was transferred to a new tube and concentrated using a SpeedVac ST:STUDY_SUMMARY until dry. Metabolites were reconstituted into 25-50 µl of water, vortexed, ST:STUDY_SUMMARY centrifuged, and transferred to vials for analysis by LCMS. LCMS was performed ST:STUDY_SUMMARY using a ZIC-pHILIC LC column coupled to a Vanquish LC and a flow gradient ST:STUDY_SUMMARY consisting of 10 mM ammonium carbonate in water and pure acetonitrile. The LC ST:STUDY_SUMMARY was coupled to an Exploris 240 mass spectrometer operated in a polarity ST:STUDY_SUMMARY switching data-dependent Top 5 mode. Full MS scan parameters for both positive ST:STUDY_SUMMARY and negative mode were set to 67-1000 m/z at a resolution of 120k and ddMS2 were ST:STUDY_SUMMARY collected at a resolution of 30k. Data files are labeled with brain, liver, and ST:STUDY_SUMMARY plasma followed by a number. The number represents a mouse. Samples 1-3 are from ST:STUDY_SUMMARY PBS-treated mice and samples 4-6 are from LOX-treated mice. ST:INSTITUTE University of British Columbia ST:DEPARTMENT Biochemistry & Molecular Biology ST:LABORATORY Parker laboratory ST:LAST_NAME Parker ST:FIRST_NAME Seth ST:ADDRESS 950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada ST:EMAIL seth.parker@bcchr.ca ST:PHONE 6048753121 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 10-17 week SU:GENDER Female SU:ANIMAL_FEED 0.9% w/w lysine, 18 ppm pyridoxine (standard diets) #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 - Plasma_PBS_1 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Plasma Sex=F; Age (days)=114; RAW_FILE_NAME(Raw file name)=ATQ_LOX_plasma_sp1.RAW SUBJECT_SAMPLE_FACTORS - Plasma_PBS_2 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Plasma Sex=F; Age (days)=73; RAW_FILE_NAME(Raw file name)=ATQ_LOX_plasma_sp2.RAW SUBJECT_SAMPLE_FACTORS - Plasma_PBS_3 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Plasma Sex=M; Age (days)=101; RAW_FILE_NAME(Raw file name)=ATQ_LOX_plasma_sp3.RAW SUBJECT_SAMPLE_FACTORS - Plasma_LOX_1 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Plasma Sex=F; Age (days)=101; RAW_FILE_NAME(Raw file name)=ATQ_LOX_plasma_sp4.RAW SUBJECT_SAMPLE_FACTORS - Plasma_LOX_2 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Plasma Sex=F; Age (days)=101; RAW_FILE_NAME(Raw file name)=ATQ_LOX_plasma_hemolysis_sp5.RAW SUBJECT_SAMPLE_FACTORS - Plasma_LOX_3 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Plasma Sex=M; Age (days)=98; RAW_FILE_NAME(Raw file name)=ATQ_LOX_plasma_sp6.RAW SUBJECT_SAMPLE_FACTORS - Brain_PBS_1 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Brain Sex=F; Age (days)=114; RAW_FILE_NAME(Raw file name)=Brain_LOX_sp1.RAW SUBJECT_SAMPLE_FACTORS - Brain_PBS_2 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Brain Sex=F; Age (days)=73; RAW_FILE_NAME(Raw file name)=Brain_LOX_sp2.RAW SUBJECT_SAMPLE_FACTORS - Brain_PBS_3 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Brain Sex=M; Age (days)=101; RAW_FILE_NAME(Raw file name)=Brain_LOX_sp3.RAW SUBJECT_SAMPLE_FACTORS - Brain_LOX_1 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Brain Sex=F; Age (days)=101; RAW_FILE_NAME(Raw file name)=Brain_LOX_sp4.RAW SUBJECT_SAMPLE_FACTORS - Brain_LOX_2 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Brain Sex=F; Age (days)=101; RAW_FILE_NAME(Raw file name)=Brain_LOX_sp5.RAW SUBJECT_SAMPLE_FACTORS - Brain_LOX_3 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Brain Sex=M; Age (days)=98; RAW_FILE_NAME(Raw file name)=Brain_LOX_sp6.RAW SUBJECT_SAMPLE_FACTORS - Liver_PBS_1 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Liver Sex=F; Age (days)=114; RAW_FILE_NAME(Raw file name)=Liver_LOX_sp1.RAW SUBJECT_SAMPLE_FACTORS - Liver_PBS_2 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Liver Sex=F; Age (days)=73; RAW_FILE_NAME(Raw file name)=Liver_LOX_sp2.RAW SUBJECT_SAMPLE_FACTORS - Liver_PBS_3 Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Liver Sex=M; Age (days)=101; RAW_FILE_NAME(Raw file name)=Liver_LOX_sp3.RAW SUBJECT_SAMPLE_FACTORS - Liver_LOX_1 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Liver Sex=F; Age (days)=101; RAW_FILE_NAME(Raw file name)=Liver_LOX_sp4.RAW SUBJECT_SAMPLE_FACTORS - Liver_LOX_2 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Liver Sex=F; Age (days)=101; RAW_FILE_NAME(Raw file name)=Liver_LOX_sp5.RAW SUBJECT_SAMPLE_FACTORS - Liver_LOX_3 Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Liver Sex=M; Age (days)=98; RAW_FILE_NAME(Raw file name)=Liver_LOX_sp6.RAW #COLLECTION CO:COLLECTION_SUMMARY Liver, brain, or plasma samples collected from mice treated with PBS or Lysine CO:COLLECTION_SUMMARY alpha-oxidase (150 U/kg) as a single intravenous injection. Tissues collected CO:COLLECTION_SUMMARY 7-hours after injection. CO:SAMPLE_TYPE Tissue or Plasma #TREATMENT TR:TREATMENT_SUMMARY PBS or Lysine alpha-oxidase (150 U/kg) TR:TREATMENT_ROUTE intravenous TR:TREATMENT_VEHICLE PBS #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Dried samples were reconstituted in 50 µL of HPLC-grade water. Samples were SP:SAMPLEPREP_SUMMARY vortexed for ~10 minutes, then centrifuged at 21,000 x g for 15 min at 4°C. 40 SP:SAMPLEPREP_SUMMARY microliters were transferred to LC vials containing glass inserts for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A 100% water, 10 mM Ammonium Carbonate, pH 9.0 CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 80-20%B (0-30 min), 20-20%B (30-40 minute), and 20-80%B (40-40.5 minute); the LC CH:FLOW_GRADIENT column was re-equilibrated using 80-80%B from 40.5-52 minute before subsequent CH:FLOW_GRADIENT injections CH:FLOW_RATE 100 uL/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The LC was coupled to a Thermo Scientific Exploris 240 mass spectrometer MS:MS_COMMENTS operating in heated electrospray ionization mode (HESI) for analysis. The MS:MS_COMMENTS following parameters were set for HESI: spray voltage 3.4 kV (positive) and 2 kV MS:MS_COMMENTS (negative), static spray voltage, sheath gas 25, aux gas 5, sweep gas 0.5, ion MS:MS_COMMENTS transfer tube temperature 320oC, and vaporizer temperature 75oC. The global MS:MS_COMMENTS parameters included an expected peak width of 20 seconds, mild trapping, and a MS:MS_COMMENTS default charge state of 1. A 40-min polarity switching data-dependent Top 5 MS:MS_COMMENTS method was used for positive mode and a data-dependent Top 3 method was used for MS:MS_COMMENTS negative mode. Full MS scan parameters for both positive and negative modes were MS:MS_COMMENTS set as follows: scan range 67-1000 m/z collected in profile mode, Orbitrap MS:MS_COMMENTS resolution 120,000, RF lens 70%, AGC target of 300%, and maximum injection time MS:MS_COMMENTS set to automatic. ddMS2 for positive mode were collected in centroid mode at an MS:MS_COMMENTS Orbitrap resolution of 30,000, isolation window of 1.5 m/z, an AGC target set to MS:MS_COMMENTS standard, a maximum injection time set to automatic, and a normalized collision MS:MS_COMMENTS energy set to 10%, 30%, and 80%. ddMS2 for negative mode were collected in MS:MS_COMMENTS centroid mode at an Orbitrap resolution of 30,000, isolation window of 2 m/z, an MS:MS_COMMENTS AGC target set to standard, a maximum injection time set to automatic, and a MS:MS_COMMENTS normalized collision energy set to 30%. For both positive and negative ddMS2, we MS:MS_COMMENTS applied an intensity threshold of 5e4 and a dynamic exclusion of 5 ppm for 10 MS:MS_COMMENTS seconds, excluding isotopes. A targeted selected ion monitoring (tSIM) scan was MS:MS_COMMENTS also included for pipecolate and P6C/P2C, and the retention time ranges were MS:MS_COMMENTS based on elution of authentic standards (pipecolate) or from positive samples MS:MS_COMMENTS (Aldh7a1-deficient tissues). The tSIM scan for pipecolate was collected from MS:MS_COMMENTS 8-12 minutes in negative mode at an isolation window of 4 m/z (for metabolomics) MS:MS_COMMENTS or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap MS:MS_COMMENTS resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, MS:MS_COMMENTS and collected in profile mode. The tSIM scan for P6C/P2C was collected from 6-10 MS:MS_COMMENTS minutes in positive mode at an isolation window of 4 m/z (for metabolomics) or MS:MS_COMMENTS 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap MS:MS_COMMENTS resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, MS:MS_COMMENTS and collected in profile mode. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Ion counts MS_METABOLITE_DATA_START Samples Plasma_PBS_1 Plasma_PBS_2 Plasma_PBS_3 Plasma_LOX_1 Plasma_LOX_2 Plasma_LOX_3 Brain_PBS_1 Brain_PBS_2 Brain_PBS_3 Brain_LOX_1 Brain_LOX_2 Brain_LOX_3 Liver_PBS_1 Liver_PBS_2 Liver_PBS_3 Liver_LOX_1 Liver_LOX_2 Liver_LOX_3 Factors Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Plasma Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Plasma Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Plasma Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Plasma Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Plasma Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Plasma Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Brain Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Brain Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Brain Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Brain Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Brain Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Brain Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Liver Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Liver Genotype:Aldh7a1-/- | Treatment:PBS | Sample source:Liver Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Liver Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Liver Genotype:Aldh7a1-/- | Treatment:LOX (150 U/kg, intravenous) | Sample source:Liver TRYPTOPHAN 5.57E+08 6.28E+08 4.27E+08 8.07E+08 7.70E+08 4.36E+08 2.50E+08 2.89E+08 2.81E+08 4.70E+08 4.79E+08 5.53E+08 2.14E+08 2.85E+08 1.95E+08 2.61E+08 3.76E+08 2.63E+08 ORNITHINE 1.77E+08 1.75E+08 1.17E+08 9.49E+05 6.68E+05 1.26E+06 8.28E+08 6.77E+08 5.46E+08 5.87E+08 6.73E+08 6.26E+08 5.79E+07 6.39E+07 4.98E+07 4.62E+07 6.20E+07 5.25E+07 P6C 1.83E+08 1.72E+08 1.24E+08 1.83E+07 1.16E+07 2.45E+07 4.75E+08 4.81E+08 4.72E+08 2.67E+07 2.25E+07 2.83E+07 2.71E+08 2.27E+08 3.84E+08 6.09E+07 6.13E+07 1.19E+08 PIPECOLATE 7.22E+06 8.85E+06 2.52E+07 1.65E+08 1.40E+08 2.01E+08 2.67E+07 2.70E+07 8.81E+07 6.79E+08 9.45E+08 1.66E+09 1.08E+08 1.65E+08 1.36E+08 3.08E+08 5.53E+08 4.87E+08 AMINOADIPATE 1362726.185 773026.683 374372.884 91524.101 91385.10465 56175.27851 2.90E+07 1.48E+07 5.68E+06 1.88E+06 2.23E+06 1.02E+06 1.12E+06 1.48E+06 1.20E+06 6.71E+04 1.40E+05 1.11E+05 2-OXOADIPIC ACID 1637281.56 841400.0116 264960.8764 139991.9588 91046.09705 60263.84096 7.18E+05 2.41E+05 1.59E+05 1.07E+05 9.95E+04 9.46E+04 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 P2C 7.67E+07 1.51E+06 8.65E+05 7.99E+05 1.68E+08 1.66E+08 1.37E+06 1.89E+06 3.46E+06 2.53E+07 1.25E+07 2.99E+07 7.00E+06 5.07E+06 8.29E+06 2.23E+09 2.16E+09 3.77E+09 6-OXO-PIPECOLATE 27167484.6 20463129.8 32771998.27 2095106.367 1966978.553 2139993.989 12617897.31 11333375.36 12913670.38 7768880.214 6898404.716 5210146.722 78071382.31 69183014.7 123761821.1 23492162.67 45403079.48 26290197.24 2-OPP 16677159.43 14747776.15 16972340.6 6369372.246 7372749.723 7109504.77 8968184.467 9386933.671 6854828.113 3152888.077 4224470.484 3054530.818 43145546.64 28003956.22 26677632.07 75280093.9 147383004.1 182008419.1 ALANINE / SARCOSINE 54939010.92 52401316.14 43985588.73 40625336.43 30879995.74 31029612.26 16861548.09 18192071.56 15803667.92 13797247.54 18756048.14 15122124.33 150264032.5 125178838.7 135038269.8 116895492 114668323.8 126013563.3 iso_Ala 12875932.76 13325238.69 12293441.46 12827682.13 11986763.55 13341821.42 2843015.209 2704436.11 2774701.404 3077145.862 2854370.277 2735046.84 4097364.225 5104576.516 4008591.164 3877085.925 3660448.648 3469026.393 ARGININE 682607333.3 644801434.8 700684557.5 53503246.8 56927576.02 72970771.69 1055288427 1336874612 1111475125 1504034626 2017300157 2013336985 53209630.76 67013798.65 40642213.81 58807502.03 32803024.17 29342274.67 iso_Arg 1601262531 1694720384 1613732284 1710526761 1740454887 1704907081 1843341678 1817711390 1829436289 1705419450 1690196250 1673076144 1900282415 1833452975 1922272942 1850972958 1959690100 1911049141 ASPARAGINE 89990239.29 85353177.85 83025807.77 68627671.21 53315961.43 50681013.56 52659854.31 54298378.34 51450479.38 43038981.7 49819615.09 44432003.29 77022814.76 82180814.79 67660600.3 92625307.48 92989932.11 114241124.5 iso_Asn 216020450.4 220876386.8 201499690.5 224101777.9 218519874.4 223141856.1 71735286.4 61251610.12 68164165.87 91674758.71 65474534.75 65829806.01 122448362.2 126805774.6 97704269.1 95230646.56 88640039.97 89013571.64 ASPARTATE 5673810.204 5256185.486 9217593.602 4687687.896 9304183.657 5489234.928 1279744313 1856268564 1681339110 1340251434 1726484566 1730928635 212384774.4 222112936.4 348891441.9 255473268.2 256177671.7 245403737.3 iso_Asp 199568856.5 204347149.1 185847628 209364540.9 184566869.3 201449022.1 71603227.99 66997973.7 69567080.02 80621091.14 65369759.37 70162295.3 123273224.8 131884913 131819432.8 109116508.8 95203538.45 93399675.17 CYSTINE 28954195 29288833.96 43289051.95 53128663.69 34606038.63 60996430.28 0 0 0 0 0 0 0 0 0 0 0 0 iso_CysCys 109463882.3 99571839.59 127881724.2 206518883.2 191545884.4 191782615.3 0 0 0 0 0 0 0 0 0 0 0 0 GLUTAMINE 1944927451 1939865994 2154952847 1900960091 1978337241 1737192845 6904066964 7299558383 6783694335 6779992464 7933007428 7008476231 3938782091 3783587384 6996912850 5188915248 5182830853 4725760966 iso_Gln 1021551009 1039475689 942972327.5 986189746.2 982897450.2 1000096082 333692379.9 307927384.2 338082928.6 398219411.9 284308361.3 322901499.9 463266337.4 549747192.8 374269291.1 357874665.6 313889136 362142002.6 GLUTAMATE 55028136.92 45469885.62 44809652.13 40590354.3 68801506.22 31444019.83 3170317757 3218881905 2966349244 2522162641 3325555153 2765434632 508051650.1 428551122 508764367.3 550205227.5 486711411.7 456884334.6 iso_Glu 290752020.2 305023628.3 273571727.2 275016241.5 249704320.6 292037392.9 35556610.17 34200274.21 35572812.42 43766199.31 30436040.27 35668048.93 67533599.15 87932036.38 76649197.47 62567430.34 52096645.57 52058998.78 GLYCINE 53344283.65 48377760.7 56791284.26 48868378.33 52071693.5 36251536.42 157300774.4 192841969.9 155282388 197705018.7 203564613.6 235924185.6 518606760.8 436777909 532242688.8 547421531.1 462217344.3 678851452 iso_Gly 45334877.98 44798349.51 41109029.18 48048855.39 50226118.26 44486785.93 30443795.09 28467410.88 28833290.12 33166226.23 29383046.87 28106206.06 43505671.64 46363080.81 38778558.5 39018257.9 39912147.68 33843718.77 HISTIDINE 2326779.784 4194042.927 3052661.601 2945218.748 768686.3933 2378601.493 905581.3532 1163483.213 865576.0538 852211.3843 987695.4825 761458.7651 20855685.44 19533093.12 27985379.75 20164259.2 19328640.42 19506878.07 iso_His 14446598.24 17230218.33 13351064.57 18558663.43 14841508.55 14881689.63 3034199.972 3430697.276 3942547.089 4357882.165 3404354.367 3873834.79 12440237.86 12943900.76 12800438.86 11538807.3 9373529.374 9954956.305 LEUCINE 22170455.07 27736538.03 22777513.46 24594144.97 21529905.49 25791051.14 10543963.57 14266415.88 14319882.08 10868840.52 14835863.59 15389666.07 29678328.14 31737158.43 34954543.82 43641131.94 47461549.3 69982523.83 iso_Leu 23648168.68 24217468.1 23737300.33 24470258.96 24286958.57 23134188.02 23040325.73 20448739.17 23344149.66 24149312.87 21599985.53 19913804.94 18006997.15 17241761.5 15402097.52 16082682.14 16214655.76 16733193.25 ISOLEUCINE 7770887.695 10006646.76 9089080.926 11211786.22 10171007.17 14558847.41 5634127.22 7507549.438 8300159.839 6970294.038 8949428.205 9262038.009 5280256.741 6976503.44 9941706.56 12773579.69 14482515.72 24412542.5 iso_Ile 18342744.82 18387330.93 18884630.23 20331129.26 21216480.96 25086434.01 29524906.75 26181579.07 30594508.54 34215724.53 28997964.59 28991566.05 6800057.24 7901727.354 8902648.308 8457545.485 8412896.028 11028682 LYSINE 286423488.5 283795713.4 286384980.3 476562.789 521918.6095 343339.096 222765836.1 267980460 240643523.5 40902564.98 56234709.09 55109153.9 593333504.6 575960338.7 575249559.2 42179415.3 25050864.91 61868526.97 iso_Lys 256488719.9 263335085.2 246209868.6 5321254.87 7406148.186 6468305.388 258780136.8 249685070.8 245852462.6 250485168.7 262201114.2 254015485.3 238731285.5 227976931.9 233364142.1 237666024.7 257884538.6 237008646.4 METHIONINE 288102479.7 258429849.9 232935899.4 188656162.6 161397446.5 100685373.5 327421108.9 369090799.6 297075367.8 197967968.4 302031702 149344747.2 113903032.8 113353715.3 106963166.6 125067642.3 144403901.1 190495567.7 iso_Met 577552370.5 585626668 565854274.6 592601026.1 587634411.1 608202105.5 740168195.4 748170681.8 769508019.8 801514115.6 750446276.2 779943063.8 334987288.3 374579736.2 418925712.1 413502843.2 401900825.5 473179142.5 PHENYLALANINE 584134014.2 638473793.2 482378400.1 543695316.7 470984467.9 564134310 568874928.3 718137106.1 546675662.6 538047257.6 770079809.2 667446382.3 943644371.6 962234161.4 952167373.9 1390035683 1383032437 1781211175 iso_Phe 1719634072 1719348986 1636096915 1694389449 1645218823 1700132458 2051588501 2011465176 2013730053 2084177117 1972007193 1940682734 1879821636 1912189570 1761874094 1814844114 1730001852 1593117347 PROLINE 1029862095 993217583.4 896655872.5 797150915.5 629328997.6 621094445.5 1119201607 1257428921 1057691509 1336468585 2095684590 1539387290 2696788027 2415788692 2183639500 2450563752 2804936043 3395764536 iso_Pro 1035241463 1015575788 969669797.7 1041284511 1020139983 1025467606 1231540749 1170243699 1230954516 1174392483 1057580407 1064218178 862775497.4 935755943.2 893866299.5 868171738.4 843812627 740515957 SERINE 38109269.18 35154087.06 39816638.22 27866753.64 23798711.48 19097936.28 230786332.2 271899550.7 206728851.2 202771239.3 342236046.9 208416704.8 71045712.43 70046799.28 94163743.68 84726253.58 98973289.48 91946925.49 iso_Ser 37000429.83 35531035.74 34523598.71 36355092.92 35410519.25 34915578.75 43551898.02 41739245.49 41949322.97 44127307.6 41777763.67 42022031 39932315.66 39332121.21 37402849.21 38130430.14 38843226.2 37696051.89 THREONINE 299111355.4 315764228.3 265937498.1 296578011.2 227478248.4 171795975.7 307446590.3 347277981.4 274582700 367706994.7 424016685.3 315599298.2 384241413.7 408873239.2 339628602.3 479010695.3 492179825.4 501796752.5 iso_Thr 238160525.8 274240088.5 216227440.3 250870811 243464686.8 228824480.4 148890148.2 120712907.9 136760391.6 179170559.6 120835085.1 126267509.7 213381829.8 232578193.3 186758674.2 205429400.7 183924252.9 168973009.6 TYROSINE 526416888.1 563656008.9 477108383.6 475789828.9 414016073.4 405948722.5 640298874.3 838452866.3 684376817.8 558993481.2 802406978.5 558257935.5 611219262.2 687866380.8 736191245.5 956019515.1 990189284.8 1276252302 iso_Tyr 1253572117 1256327508 1174389150 1245172977 1228430755 1226501103 1526256206 1475922929 1512099963 1613618250 1435097840 1468905473 1261274024 1318789606 1238567531 1243849551 1187587857 1147523495 VALINE 407375811.3 466965751.9 432379624.1 404772977.3 355131493.1 484054907.9 161054177.4 251674730.7 272652258.8 162128359.9 236163640.9 297474121.6 422993203.1 535370417.7 685873218.2 733613874.9 795156268.5 907541412.9 iso_Val 354320724 353310010.4 324443011.8 282720011.8 280132246.5 264455544.5 359199992 358078256.1 366046414.5 316994562.7 270381079.5 279936411.6 333412552.5 336041635.1 312031085.9 227121232.7 206145273.8 171041147.6 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Chemical Formula RT Mass (m/z) Category TRYPTOPHAN C11H12N2O2 8.33 205.0972 HMDB0013609 ORNITHINE C5H12N2O2 24 133.0972 HMDB0000214 P6C C6H9O2N 8.43 128.07061 HMDB0012130 / HMDB0001084 PIPECOLATE C6H11NO2 9.32 128.0717 HMDB0000716 AMINOADIPATE C6H11NO4 12.8 162.0761 HMDB0000510 2-OXOADIPIC ACID C6H8O5 12.51 159.0299 HMDB0000225 P2C C6H9NO2 5 128.07061 6-OXO-PIPECOLATE C6H9O3N 5 144.06529 HMDB0061705 2-OPP C9H15O3N 6.91 186.11247 na ALANINE / SARCOSINE C3H7NO2 12.48 90.055 HMDB0000161 / HMDB0000271 iso_Ala C3H7O2N 12.36 94.06183 standard1 ARGININE C6H14N4O2 27.85 175.119 HMDB0000517 iso_Arg C6H14N4O2 27.83 185.12675 standard2 ASPARAGINE C4H8N2O3 13.15 133.0608 HMDB0000168 iso_Asn C4H8N2O3 13.03 139.06784 standard3 ASPARTATE C4H7NO4 12.55 132.0302 HMDB0000191 iso_Asp C4H7NO4 12.85 137.04037 standard4 CYSTINE C6H12N2O4S2 14.38 241.0317 HMDB0000192 iso_CysCys C6H12N2O4S2 14.22 249.04469 standard5 GLUTAMINE C5H10N2O3 12.98 147.0764 HMDB0000641 iso_Gln C5H10N2O3 12.8 154.08685 standard6 GLUTAMATE C5H9NO4 12.43 148.0604 HMDB0000148 iso_Glu C5H9NO4 12.45 154.07376 standard7 GLYCINE C2H5NO2 13.51 76.0393 HMDB0000123 iso_Gly C2H5O2N 13.45 79.04284 standard8 HISTIDINE C6H9N3O2 12.4 156.0773 HMDB0000177 iso_His C6H9N3O2 12.25 165.0876 standard9 LEUCINE C6H13NO2 7.39 132.1019 HMDB0000687 iso_Leu C6H13O2N 7.31 139.11873 standard11 ISOLEUCINE C6H13NO2 8 132.1019 HMDB0000172 iso_Ile C6H13O2N 8 139.11879 standard10 LYSINE C6H14N2O2 26.05 147.1126 HMDB0000182 iso_Lys C6H14N2O2 26.16 155.12656 standard12 METHIONINE C5H11NO2S 8.29 150.0583 HMDB0000696 iso_Met C5H11NO2S 8.25 156.07175 standard13 PHENYLALANINE C9H11NO2 6.48 166.0863 HMDB0000159 iso_Phe C9H11NO2 6.42 176.11308 standard14 PROLINE C5H9NO2 9.83 116.0706 HMDB0000162 iso_Pro C5H9NO2 9.82 122.08415 standard15 SERINE C3H7NO3 13.71 104.0353 HMDB0000187 iso_Ser C3H7O3N 13.6 108.04213 standard16 THREONINE C4H9NO3 12.43 120.0655 HMDB0000167 iso_Thr C4H9NO3 11.8 125.07566 standard17 TYROSINE C9H11NO3 10.17 182.0812 HMDB0000158 iso_Tyr C9H11NO3 10.05 192.10793 standard18 VALINE C5H11NO2 9.53 118.0863 HMDB0000883 iso_Val C5H11O2N 9.45 124.09978 standard19 METABOLITES_END #END