#METABOLOMICS WORKBENCH xiachaoyi_834_20240926_180859 DATATRACK_ID:5233 STUDY_ID:ST003504 ANALYSIS_ID:AN005752 PROJECT_ID:PR002150 VERSION 1 CREATED_ON September 28, 2024, 8:37 pm #PROJECT PR:PROJECT_TITLE Methionine-SAM metabolism-dependent ubiquinone synthesis is crucial for ROS PR:PROJECT_TITLE accumulation in ferroptosis induction PR:PROJECT_SUMMARY Ferroptosis is a cell death modality in which iron-dependent lipid peroxides PR:PROJECT_SUMMARY accumulate on cell membranes. Cysteine, a limiting substrate for the glutathione PR:PROJECT_SUMMARY system that neutralizes lipid peroxidation and prevents ferroptosis, can be PR:PROJECT_SUMMARY converted by cystine reduction or synthesized from methionine. However, PR:PROJECT_SUMMARY accumulating evidence shows methionine-based cysteine synthesis fails to PR:PROJECT_SUMMARY effectively rescue intracellular cysteine levels upon cystine deprivation and is PR:PROJECT_SUMMARY unable to inhibit ferroptosis. Here, we report that methionine-based cysteine PR:PROJECT_SUMMARY synthesis is tissue-specific. Unexpectedly, we find that rather than inhibiting PR:PROJECT_SUMMARY ferroptosis, methionine in fact plays an essential role during cystine PR:PROJECT_SUMMARY deprivation-induced ferroptosis. Methionine-derived S-adenosylmethionine (SAM) PR:PROJECT_SUMMARY contributes to methylation-dependent ubiquinone synthesis, which leads to lipid PR:PROJECT_SUMMARY peroxides accumulation and subsequent ferroptosis. Moreover, SAM supplementation PR:PROJECT_SUMMARY synergizes with imidazole ketone erastin in a tumor growth suppression mouse PR:PROJECT_SUMMARY model. Inhibiting the enzyme that converts methionine to SAM protects heart PR:PROJECT_SUMMARY tissue from doxorubicin-induced and ferroptosis-driven cardiomyopathy. This PR:PROJECT_SUMMARY study broadens our understanding about the intersection of amino acid metabolism PR:PROJECT_SUMMARY and ferroptosis regulation, providing insight into the underlying mechanisms and PR:PROJECT_SUMMARY suggesting the methionine-SAM axis is a promising therapeutic strategy to treat PR:PROJECT_SUMMARY ferroptosis-related diseases. PR:INSTITUTE Northeast Normal University PR:LAST_NAME xia PR:FIRST_NAME chaoyi PR:ADDRESS 5268 Renmin Street, changchun, ji lin, 130024, China PR:EMAIL xiacy834@nenu.edu.cn PR:PHONE 18166850252 #STUDY ST:STUDY_TITLE Methionine-SAM metabolism-dependent ubiquinone synthesis is crucial for ROS ST:STUDY_TITLE accumulation in ferroptosis induction ST:STUDY_SUMMARY Ferroptosis is a cell death modality in which iron-dependent lipid peroxides ST:STUDY_SUMMARY accumulate on cell membranes. Cysteine, a limiting substrate for the glutathione ST:STUDY_SUMMARY system that neutralizes lipid peroxidation and prevents ferroptosis, can be ST:STUDY_SUMMARY converted by cystine reduction or synthesized from methionine. However, ST:STUDY_SUMMARY accumulating evidence shows methionine-based cysteine synthesis fails to ST:STUDY_SUMMARY effectively rescue intracellular cysteine levels upon cystine deprivation and is ST:STUDY_SUMMARY unable to inhibit ferroptosis. Here, we report that methionine-based cysteine ST:STUDY_SUMMARY synthesis is tissue-specific. Unexpectedly, we find that rather than inhibiting ST:STUDY_SUMMARY ferroptosis, methionine in fact plays an essential role during cystine ST:STUDY_SUMMARY deprivation-induced ferroptosis. Methionine-derived S-adenosylmethionine (SAM) ST:STUDY_SUMMARY contributes to methylation-dependent ubiquinone synthesis, which leads to lipid ST:STUDY_SUMMARY peroxides accumulation and subsequent ferroptosis. Moreover, SAM supplementation ST:STUDY_SUMMARY synergizes with imidazole ketone erastin in a tumor growth suppression mouse ST:STUDY_SUMMARY model. Inhibiting the enzyme that converts methionine to SAM protects heart ST:STUDY_SUMMARY tissue from doxorubicin-induced and ferroptosis-driven cardiomyopathy. This ST:STUDY_SUMMARY study broadens our understanding about the intersection of amino acid metabolism ST:STUDY_SUMMARY and ferroptosis regulation, providing insight into the underlying mechanisms and ST:STUDY_SUMMARY suggesting the methionine-SAM axis is a promising therapeutic strategy to treat ST:STUDY_SUMMARY ferroptosis-related diseases. To test, we firstly performed a metabolomics ST:STUDY_SUMMARY analysis of HT1080 cells that were cultured in medium lacking cystine or lacking ST:STUDY_SUMMARY both cystine and methionine. The metabolomics results containing full profile of ST:STUDY_SUMMARY the metabolites was subjected to cluster assay, and the triplicates of each ST:STUDY_SUMMARY group were automatically clustered together, indicating better homogeneity of ST:STUDY_SUMMARY the data in each condition. Then, we filtrated the metabolomics data with the ST:STUDY_SUMMARY threshold fold change (FC) ≥2 and P≤ 0.05.Only the RP Pos method results are ST:STUDY_SUMMARY used/reported in this study. The results showed that the cellular cysteine and ST:STUDY_SUMMARY GSH levels were expectedly depressed upon cystine limitation; meanwhile, once ST:STUDY_SUMMARY methionine was restricted, cellular SAM level was significantly reduced. This ST:STUDY_SUMMARY highlights the decisive regulatory role of methionine on the metabolite levels ST:STUDY_SUMMARY of SAM. ST:INSTITUTE Northeast Normal University ST:LAST_NAME xia ST:FIRST_NAME chaoyi ST:ADDRESS 5268 Renmin Street, changchun, ji lin, 130024, China ST:EMAIL xiacy834@nenu.edu.cn ST:PHONE 18166850252 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male #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 +CC+Met_2 XiaChaoYi_A4_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:+CC+Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A4_RP pos.mzML SUBJECT_SAMPLE_FACTORS -CC+Met_3 XiaChaoYi_A5_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:-CC+Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A5_RP pos.mzML SUBJECT_SAMPLE_FACTORS -CC-Met_1 XiaChaoYi_A6_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:-CC-Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A6_RP pos.mzML SUBJECT_SAMPLE_FACTORS +CC+Met_1 XiaChaoYi_A7_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:+CC+Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A7_RP pos.mzML SUBJECT_SAMPLE_FACTORS -CC+Met_2 XiaChaoYi_A8_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:-CC+Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A8_RP pos.mzML SUBJECT_SAMPLE_FACTORS -CC-Met_3 XiaChaoYi_A9_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:-CC-Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A9_RP pos.mzML SUBJECT_SAMPLE_FACTORS +CC+Met_3 XiaChaoYi_A10_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:+CC+Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A10_RP pos.mzML SUBJECT_SAMPLE_FACTORS -CC+Met_1 XiaChaoYi_A11_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:-CC+Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A11_RP pos.mzML SUBJECT_SAMPLE_FACTORS -CC-Met_2 XiaChaoYi_A12_RP pos Sample source:HT1080 human epithelial cancer cells | Treatment:-CC-Met Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=XiaChaoYi_A12_RP pos.mzML #COLLECTION CO:COLLECTION_SUMMARY Cells were seeded at a density of 2×106 per well in a 10 cm glass bottom cell CO:COLLECTION_SUMMARY culture dish and grown for 12 h in DMEM supplemented with 10% Fetal Bovine CO:COLLECTION_SUMMARY Serum（FBS）, 1% Penicillin-Streptomycin(P/S). CO:COLLECTION_PROTOCOL_FILENAME 2024-NC.pdf CO:SAMPLE_TYPE HT1080 Human epithelial cancer cells #TREATMENT TR:TREATMENT_SUMMARY HT1080 cells were cultured in the medium ± cystine or ± methionine as TR:TREATMENT_SUMMARY indicated for 10 h. The metabolites levels were measured by UHPLC-HRMS. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were trypsinized, washed and resuspended in methanol: acetonitrile: ddH2O2 SP:SAMPLEPREP_SUMMARY (2:2:1, v/v) after indicated treatment. Then subjected to LC/MS analysis at SP:SAMPLEPREP_SUMMARY LipidALL Technologies (Changzhou, China). Briefly, polar metabolites were SP:SAMPLEPREP_SUMMARY extracted using 1000 µl of ice-cold methanol: H2O (4:1, v/v), and incubated at SP:SAMPLEPREP_SUMMARY 2000 × g for 30 min at 4oC. At the end of the incubation, samples were SP:SAMPLEPREP_SUMMARY centrifuged for 10 min at 13,000 × g at 4oC. Clean supernatant was transferred SP:SAMPLEPREP_SUMMARY to a new tube. Extracts were dried in a centrifugal concentrator. SP:SAMPLEPREP_PROTOCOL_FILENAME 2024-NC.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0 - 1.0 min with 2% B, 1.0 - 6.0 min with 2% - 42% B, 6.0 - 8.0 min with 42% - CH:FLOW_GRADIENT 65% B, 8.0 - 10.0 min with 65% - 76% B, 10.0 - 11.0 min with 76% - 100% B, 11.0 CH:FLOW_GRADIENT - 14.0 min with 100% - 100% B. CH:FLOW_RATE 0.35 mL/min CH:COLUMN_TEMPERATURE 40 CH:METHODS_FILENAME 2024-NC.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE 2024-NC.pdf #MS MS:INSTRUMENT_NAME ABI Sciex 5600+ TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS parameters for detection were: ESI source voltage positive ion mode 5.5 kV, MS:MS_COMMENTS negative ion mode -4.5 kV; vaporizer temperature, 450°C; drying gas (N2) MS:MS_COMMENTS pressure, 50 psi; nebulizer gas (N2) pressure, 50 psi; curtain gas (N2) MS:MS_COMMENTS pressure, 35 psi; The scan range was m/z 60-800. Information-dependent MS:MS_COMMENTS acquisition mode was used for MS/MS analyses of the metabolites. Collision MS:MS_COMMENTS energy was set at (±) 35 ± 15 eV. Data acquisition and processing were MS:MS_COMMENTS performed using Analyst® TF 1.7.1 Software (AB Sciex, Concord, ON, Canada). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples XiaChaoYi_A7_RP pos XiaChaoYi_A4_RP pos XiaChaoYi_A10_RP pos XiaChaoYi_A11_RP pos XiaChaoYi_A8_RP pos XiaChaoYi_A5_RP pos XiaChaoYi_A6_RP pos XiaChaoYi_A12_RP pos XiaChaoYi_A9_RP pos Factors Sample source:HT1080 human epithelial cancer cells | Treatment:+CC+Met Sample source:HT1080 human epithelial cancer cells | Treatment:+CC+Met Sample source:HT1080 human epithelial cancer cells | Treatment:+CC+Met Sample source:HT1080 human epithelial cancer cells | Treatment:-CC+Met Sample source:HT1080 human epithelial cancer cells | Treatment:-CC+Met Sample source:HT1080 human epithelial cancer cells | Treatment:-CC+Met Sample source:HT1080 human epithelial cancer cells | Treatment:-CC-Met Sample source:HT1080 human epithelial cancer cells | Treatment:-CC-Met Sample source:HT1080 human epithelial cancer cells | Treatment:-CC-Met Butyrylcarnitine 26427.9476 27155.77889 22194.91525 23267.92453 25663.7931 27419.64286 30495.41284 32775 32104 Dodecanoylcarnitine 2872.489083 4478.39196 2461.864407 1956.981132 2376.724138 2844.642857 3096.330275 3588.333333 2982.4 Isobutyrylcarnitine 26323.1441 27125.62814 22127.11864 23207.54717 25603.44828 27375 30440.36697 32691.66667 32040 Isovalerylcarnitine 15013.10044 16120.60302 15144.0678 8800 10956.89655 10401.78571 7363.302752 7845.833333 7272 L-Acetylcarnitine 201572.0524 223618.0905 152033.8983 143320.7547 191465.5172 195089.2857 212201.8349 201416.6667 187440 Pivaloylcarnitine 5383.406114 5521.60804 5897.457627 4171.320755 3910.344828 4048.214286 3550.458716 4544.166667 3487.2 Propionylcarnitine 34628.82096 46080.40201 31144.0678 14098.11321 19000 26830.35714 26110.09174 15883.33333 18192 Valerylcarnitine 5406.113537 5506.532663 5846.610169 4141.132075 3862.068966 3971.428571 3577.981651 4525.833333 3457.6 Methacholine 1932.751092 2700.502513 1361.016949 1860.377358 1798.275862 2332.142857 2336.697248 1895 2648 Pantothenic acid 2908296.943 3169849.246 2531355.932 2212075.472 3006034.483 3371428.571 2594495.413 2185833.333 2496000 N-Acetylglutamine 1295196.507 4466331.658 1046610.169 1348679.245 6200000 1682142.857 2343119.266 5820833.333 1465600 1-Methylhistidine 4937.117904 5539.698492 5310.169492 6084.528302 5708.62069 6709.821429 6650.458716 6986.666667 6651.2 Betaine 196282.0134 216544.8474 170109.8901 170828.2627 193309.5038 210114.6597 296054.8146 300660.9773 267836.8887 Citrulline 563527.5392 574776.3774 450969.0143 461842.4207 597254.8602 605091.0638 746363.1332 645937.0316 631142.0225 Creatine 4011877.683 4369179.447 3148004.661 3528434.614 4326934.183 4895733.115 6957643.995 5901088.892 6207185.811 Creatinine 31330.45847 39731.69917 25798.31622 27041.60992 32143.55158 35108.38218 42990.48924 36516.82157 32828.36336 DL-2-Aminooctanoic acid 810.2183406 1322.613065 892.3728814 1480.754717 1555.172414 1459.821429 1603.669725 1341.666667 1362.4 L-Glutamic acid 11811000.17 11284191.91 9349043.738 10571937.78 12017407.14 14340025.05 8700825.434 8225454.645 11120374.71 gamma-Aminobutyric acid 6615.720524 5254.271357 4611.864407 6476.981132 7475.862069 6940.178571 8649.541284 9233.333333 8968 Glycine 3413.973799 3626.130653 2892.372881 3060.377358 8022.413793 3784.821429 5296.330275 10491.66667 7100.8 Isovalerylglycine 4000.873362 3752.763819 3830.508475 4031.698113 5487.068966 4702.678571 5096.330275 5350.833333 6114.4 L-2-Amino-4-methylenepentanedioic acid 1390.393013 1640.201005 1639.830508 1061.886792 1184.482759 1699.107143 1029.357798 1159.166667 1787.2 L-Alanine 69414.84716 54773.86935 61881.35593 112301.8868 128189.6552 113571.4286 137889.9083 172750 172560 L-Arginine 111251.304 138100.2661 106949.8277 87780.05974 124411.2677 142204.0128 162157.3779 137706.1802 127626.8821 L-Asparagine 16218.34061 14522.61307 15737.28814 18037.73585 24060.34483 25008.92857 25137.61468 27258.33333 24984 L-Aspartic acid 248122.2707 228844.2211 216016.9492 214566.0377 281551.7241 294732.1429 274036.6972 263166.6667 312240 Leucyl-Aspartate 50716.15721 53608.0402 47864.40678 847547.1698 1000000 1037500 1045871.56 898333.3333 942400 Leucylproline 11371.17904 13276.38191 9771.186441 11479.24528 14215.51724 16044.64286 18899.08257 15950 17816 L-gamma-glutamyl-L-isoleucine 211877.7293 221809.0452 190338.9831 1746415.094 1943965.517 2235714.286 2055045.872 1733333.333 1913600 L-gamma-glutamyl-L-leucine 103493.4498 95788.94472 96694.91525 1528301.887 1593103.448 1914285.714 1673394.495 1545000 1685600 L-Glutamine 695871.8463 762233.9309 445057.0157 1807644.388 2341397.978 2501841.033 2787135.459 2521426.619 2817445.568 L-Histidine 57848.29855 66316.04735 47106.30055 68253.96906 98665.27721 83688.89822 109641.6496 80073.89465 84136.09033 L-Isoleucine 7966817.197 8859422.374 6674536.69 9221192.497 10787363.18 11480514.58 11435656.92 10268364.04 10593337.69 L-Leucine 7944486.748 8839062.906 6654933.375 9191708.268 10761820.88 11452536.08 11407651.23 10243400.43 10569550.25 L-Lysine 138898.5396 150948.9799 118096.6288 147110.4182 179558.8804 183989.3852 192962.6374 179413.1496 171957.8802 L-Methionine 4209061.963 4522377.48 3118426.05 5683635.282 6827701.413 7483965.86 50920.66543 45252.60117 48435.84295 L-Phenylalanine 14255424.34 15448532.54 11749786.83 16288929.56 19950789.08 21606298.02 20139847.38 17135638.9 17922140.73 L-Proline 1052361.638 1152056.512 915741.7107 1275952.094 1555875.598 1707404.647 2111988.228 1823801.151 1813842.966 L-Threonine 310375.2582 328613.1644 267694.3779 402977.6727 471565.4847 558107.7204 741285.5573 674797.9105 751425.7545 L-Tryptophan 12010998.71 13267015.73 10216755.48 13649856.1 16569274.93 17521448.29 17275472.73 16104895.11 16250900.79 L-Tyrosine 9624804.534 11238823.06 8142558.843 11468393.11 13163319.44 14579015.44 13745788.59 12460542.93 12421197.2 L-Valine 1367906.496 1488246.878 1106876.725 1528359.23 1812114.408 2002950.336 1976264.073 1751997.459 1778765.98 Methionine sulfoxide 11755.45852 11175.8794 9610.169492 12626.41509 12672.41379 14919.64286 6857.798165 6715 7991.2 N2-Acetylornithine 26131.00437 22864.32161 21864.40678 24732.07547 14025.86207 30080.35714 27853.21101 12933.33333 27120 N6,N6,N6-Trimethyl-L-lysine 7103.930131 6929.648241 7929.661017 6516.226415 6832.758621 6575.892857 6528.440367 6738.333333 5310.4 Ornithine 12244.54148 11557.78894 11771.18644 18075.4717 21931.03448 19375 19587.15596 25066.66667 22536 Phenylalanylphenylalanine 1357.20524 1643.21608 1505.084746 1992.45283 2275 2088.392857 1762.385321 2650 1800.8 Pipecolic acid 27117.90393 21075.37688 25881.35593 21101.88679 11284.48276 26214.28571 21706.42202 9108.333333 23376 Proline betaine 134934.4978 156482.4121 110932.2034 108981.1321 130172.4138 138839.2857 176146.789 135166.6667 157680 Prolylhydroxyproline 41589.51965 41246.23116 37474.57627 23426.41509 11612.06897 28964.28571 36981.65138 14508.33333 30712 Pyroglutamic acid 981659.3886 1231155.779 883898.3051 1347169.811 1743103.448 1677678.571 2171559.633 1892500 1866400 Pyroglutamine 40969.43231 46502.51256 34381.35593 39350.9434 52974.13793 51776.78571 66568.80734 67083.33333 77104 S-Methyl-L-cysteinesulfoxide 157292.5764 198291.4573 158898.3051 181132.0755 208362.069 214910.7143 222385.3211 194416.6667 169200 Symmetric dimethylarginine 31467.24891 35206.03015 31000 29547.16981 30517.24138 37098.21429 32311.92661 24083.33333 30056 Taurine 70340.30631 97118.40232 66099.59337 43945.09334 44300.2299 41041.06773 76023.4841 48709.05299 54911.54833 4-Hydroxyproline 37536.59222 43951.30007 33720.58803 52981.24191 69117.35866 73232.75326 72977.4911 69633.95539 81984.81988 L-Cystathionine 6193.0131 4491.457286 7283.050847 7009.056604 7631.896552 8725.892857 359.1743119 330.4166667 482.88 L-Cysteine 62253.27511 47567.8392 52152.54237 3378.113208 2004.310345 3392.857143 4075.229358 2223.333333 5644 Glutathione 9484716.157 9899497.487 8728813.559 429358.4906 501724.1379 505625 1641284.404 1266666.667 1490400 L-homocysteine 3331.877729 2933.668342 3135.59322 2554.716981 420.4310345 2538.392857 3722.018349 89.41666667 3517.6 Phenacemide 12567.68559 16633.16583 16245.76271 10769.81132 11646.55172 11982.14286 15376.14679 21366.66667 13016 Dihydroactinidiolide 51493.44978 55286.43216 46228.81356 41245.28302 44603.44828 53098.21429 51183.48624 43400 44856 2-Aminobenzoic acid 48096.06987 51457.28643 45830.50847 44007.54717 41146.55172 50160.71429 53688.07339 38541.66667 41440 Hippuric acid 3158.453009 4511.698606 3323.111705 2521.007478 2919.187471 2767.947864 2954.299305 2378.756659 2750.172259 1-(beta-D-Ribofuranosyl)-1,4-dihydronicotinamide 9528.384279 11748.74372 8084.745763 7947.169811 9258.62069 8747.321429 10238.53211 7665 7663.2 2-Deoxygalactopyranose 6464.628821 6919.59799 6174.576271 5553.962264 4893.103448 4614.285714 8577.06422 8242.5 7369.6 D-Fructofuranose 1,2':2,3'-dianhydride 9283.842795 5103.517588 4205.084746 3513.962264 3817.241379 4146.428571 4331.192661 7615.833333 5741.6 Trehalose 7241.048035 5439.19598 3497.457627 2886.792453 3992.241379 3967.857143 5718.348624 5256.666667 5140 4-Trimethylammoniobutanoic acid 22864.62882 12804.0201 26322.0339 18898.11321 7368.965517 20633.92857 16623.85321 7300.833333 16312 Glycerophosphocholine 8104803.493 7641206.03 7023728.814 8264150.943 9241379.31 9026785.714 13596330.28 13908333.33 15144000 LysoPC(14:0) 83764.19214 135276.3819 85000 60173.58491 182327.5862 84303.57143 103394.4954 138916.6667 92880 LysoPC(16:0) 286986.8996 469145.7286 292033.8983 203698.1132 598620.6897 347946.4286 388440.367 457916.6667 338240 LysoPC(P-16:0/0:0) 220436.6812 45577.88945 24991.52542 39215.09434 330000 18133.92857 176238.5321 251250 296640 PC (16:0) 14096.06987 24231.15578 16254.23729 10883.01887 25172.41379 18446.42857 21394.49541 18600 22424 LysoPE(16:0) 61956.33188 96512.56281 66754.23729 37796.22642 90603.44828 58830.35714 62284.40367 60708.33333 62096 LysoPE(18:1) 114410.4803 251457.2864 127966.1017 86641.50943 313620.6897 160714.2857 170825.6881 191250 144800 LysoPE(18:2) 17860.26201 31055.27638 18889.83051 13750.9434 61387.93103 20169.64286 17385.3211 28433.33333 20424 SAH 681834.0611 1315577.889 613728.8136 558792.4528 2050862.069 680357.1429 339724.7706 428916.6667 304000 5'-Methylthioadenosine 3048908.297 4801005.025 2168644.068 2382641.509 3981896.552 2991964.286 1136697.248 645000 665280 SAM 150131.0044 84432.1608 159576.2712 154943.3962 58724.13793 175089.2857 16449.54128 5620 20776 2-Acetylpyrazine 1274235.808 1452261.307 1175423.729 1098867.925 1269827.586 1346428.571 1698165.138 1416666.667 1461600 L-Kynurenine 64873.36245 61527.63819 58093.22034 57607.54717 62534.48276 57321.42857 67651.37615 59158.33333 61056 2-Piperidinone 3613.100437 5028.140704 3229.661017 3232.45283 3641.37931 3826.785714 3750.458716 3329.166667 2956.8 Lumichrome 50620.08734 51467.33668 45838.98305 36626.41509 38870.68966 46750 44385.3211 30658.33333 45408 1-Methyladenosine 28008.73362 30743.71859 28237.28814 58347.16981 71129.31034 65955.35714 63917.43119 59333.33333 56104 Adenosine 1665502.183 741507.5377 1794915.254 1452075.472 1087931.034 1132142.857 927522.9358 1128333.333 1404000 N2,N2-Dimethylguanosine 4498.689956 5188.944724 4732.20339 3328.301887 3111.206897 3063.392857 2945.87156 2961.666667 2572.8 Succinyladenosine 29082.96943 23236.1809 30389.83051 27743.39623 23646.55172 21714.28571 20284.40367 19183.33333 20120 Guanosine monophosphate 536157.2052 629849.2462 501355.9322 654867.9245 629568.9655 616250 763944.9541 583166.6667 510400 1-Methylguanine 86515.28384 112763.8191 79449.15254 86641.50943 99741.37931 104642.8571 126330.2752 102583.3333 108800 Allopurinol 55790.39301 54884.42211 54737.28814 62316.98113 66094.82759 68892.85714 72504.58716 59275 63432 Guanine 110655.0218 93447.23618 110932.2034 100075.4717 103965.5172 104107.1429 110091.7431 99500 109840 Hypoxanthine 491309.9979 455526.6501 465073.7033 497853.3361 467155.1505 496532.0722 453046.1988 421347.3867 381304.9173 1-methylnicotinamide 10882.09607 3192.964824 8788.135593 8845.283019 6275.862069 9151.785714 15614.6789 12475 4948 2-Methylnicotinamide 43502.18341 24874.37186 37000 31501.88679 15905.17241 38107.14286 38311.92661 12708.33333 37032 N1-Methyl-2-pyridone-5-carboxamide 7175.545852 6025.125628 7000.847458 6414.339623 6725 6374.107143 6578.899083 6551.666667 6941.6 Niacinamide 1275109.17 1453266.332 1176271.186 1099622.642 1270689.655 1347321.429 1699082.569 1418333.333 1463200 Cytidine 73318.77729 79567.8392 73449.15254 70694.33962 80353.44828 82214.28571 89091.74312 72633.33333 78776 CDP-ethanolamine 98427.9476 96160.80402 89661.01695 91924.5283 106810.3448 122053.5714 135596.3303 117500 157680 Cytosine 202008.7336 263115.5779 213983.0508 201735.8491 215431.0345 218750 259082.5688 202750 215600 Thymine 8925.764192 8006.030151 9127.118644 8083.018868 8185.344828 7494.642857 6859.633028 7240 6730.4 Uracil 74698.68996 82743.71859 83889.83051 68777.35849 74284.48276 71946.42857 76238.53211 66341.66667 62976 Pyrrolidine 461222.7074 539095.4774 422372.8814 581283.0189 679224.1379 658571.4286 687706.422 683500 683040 Acetylcholine 97117.90393 109949.7487 78161.01695 84150.9434 97758.62069 105535.7143 96513.76147 79766.66667 90160 Choline 549828.2336 511036.6508 547356.0304 658272.4725 779897.618 788050.3461 1112820.063 1099878.788 1146153.046 L-Carnitine 72873.36245 87075.37688 67186.44068 62958.49057 76637.93103 81089.28571 109816.5138 85166.66667 91120 Phosphorylcholine 2096943.231 1934673.367 1691525.424 1512452.83 2015517.241 2042857.143 2071559.633 2007500 2541600 3-methyl-2-Quinoxalinone 66043.66812 96492.46231 93220.33898 56415.09434 65163.7931 69026.78571 88458.7156 118333.3333 78448 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Experimental m/z Retention time (min) Database ID Formula Butyrylcarnitine 232.154 4.72 HMDB0002013 C11H21NO4 Dodecanoylcarnitine 153.0901 1.37 HMDB0002250 C19H37NO4 Isobutyrylcarnitine 232.1537 4.64 HMDB0000736 C11H21NO4 Isovalerylcarnitine 246.1713 5.35 HMDB0000688 C12H23NO4 L-Acetylcarnitine 204.1233 2.27 HMDB0000201 C9H17NO4 Pivaloylcarnitine 246.1705 5.47 HMDB0041993 C12H23NO4 Propionylcarnitine 218.1376 3.87 HMDB0000824 C10H19NO4 Valerylcarnitine 246.1701 5.47 HMDB0013128 C12H23NO4 Methacholine 160.1321 1.89 HMDB0015654 C8H17NO2 Pantothenic acid 220.1181 4.5 HMDB0000210 C9H17NO5 N-Acetylglutamine 189.087 1.54 HMDB0006029 C7H12N2O4 1-Methylhistidine 170.0932 1.29 HMDB0000001 C7H11N3O2 Betaine 118.0855 1.41 HMDB00043 C5H11NO2 Citrulline 176.1026 1.42 HMDB00904 C6H13N3O3 Creatine 132.0742 1.51 HMDB00064 C4H9N3O2 Creatinine 114.0647 1.49 HMDB00562 C4H7N3O DL-2-Aminooctanoic acid 160.1328 5.73 HMDB0000991 C8H17NO2 L-Glutamic acid 148.0606 1.41 HMDB00148 C5H9NO4 gamma-Aminobutyric acid 344.2805 9.18 HMDB0000112 C4H9NO2 Glycine 76.0386 1.31 HMDB0000123 C2H5NO2 Isovalerylglycine 160.0964 1.47 HMDB0000678 C7H13NO3 L-2-Amino-4-methylenepentanedioic acid 160.0607 1.57 HMDB0029433 C6H9NO4 L-Alanine 90.0543 1.36 HMDB0000161 C3H7NO2 L-Arginine 175.1179 1.29 HMDB0000517 C6H14N4O2 L-Asparagine 133.0594 1.32 HMDB0000168 C4H8N2O3 L-Aspartic acid 134.0448 1.32 HMDB0000191 C4H7NO4 Leucyl-Aspartate 247.1277 4.2 HMDB0028925 C10H18N2O5 Leucylproline 229.1541 2.36 HMDB0011175 C11H20N2O3 L-gamma-glutamyl-L-isoleucine 261.1436 4.8 HMDB0011170 C11H20N2O5 L-gamma-glutamyl-L-leucine 261.1436 5 HMDB0011171 C11H20N2O5 L-Glutamine 147.0765 1.32 HMDB0000641 C5H10N2O3 L-Histidine 156.0762 1.29 HMDB0000177 C6H9N3O2 L-Isoleucine 132.1019 3.38 HMDB0000172 C6H13NO2 L-Leucine 132.1012 3.64 HMDB0000687 C6H13NO2 L-Lysine 147.1125 1.18 HMDB0000182 C6H14N2O2 L-Methionine 150.0578 2.27 HMDB0000696 C5H11NO2S L-Phenylalanine 166.0856 4.34 HMDB0000159 C9H11NO2 L-Proline 116.0706 1.54 HMDB0000162 C5H9NO2 L-Threonine 120.0649 1.37 HMDB0000167 C4H9NO3 L-Tryptophan 205.0957 4.95 HMDB0000929 C11H12N2O2 L-Tyrosine 182.0809 3.57 HMDB0000158 C9H11NO3 L-Valine 118.0855 1.92 HMDB0000883 C5H11NO2 Methionine sulfoxide 166.0525 1.41 HMDB0002005 C5H11NO3S N2-Acetylornithine 175.1071 1.62 HMDB0003357 C7H14N2O3 N6,N6,N6-Trimethyl-L-lysine 189.1341 1.31 HMDB0001325 C9H20N2O2 Ornithine 133.0964 1.17 HMDB0000214 C5H12N2O2 Phenylalanylphenylalanine 313.1537 6.18 HMDB0013302 C18H20N2O3 Pipecolic acid 130.085 2.16 HMDB0000070 C6H11NO2 Proline betaine 144.1006 1.59 HMDB0004827 C7H13NO2 Prolylhydroxyproline 229.1173 1.69 HMDB0006695 C10H16N2O4 Pyroglutamic acid 130.0498 2.66 HMDB0000267 C5H7NO3 Pyroglutamine 129.0656 1.46 HMDB0062558 C5H8N2O2 S-Methyl-L-cysteinesulfoxide 152.0313 1.33 HMDB0029432 C4H9NO3S Symmetric dimethylarginine 203.1517 1.49 HMDB0001539 C8H18N4O2 Taurine 126.0214 1.33 HMDB0000251 C2H7NO3S 4-Hydroxyproline 132.0611 1.37 HMDB0000725 C5H9NO3 L-Cystathionine 223.0738 1.37 HMDB0000099 C7H14N2O4S L-Cysteine 122.0271 1.44 HMDB0000574 C3H7NO2S Glutathione 104.0691 1.33 HMDB0000125 C10H17N3O6S L-homocysteine 136.0426 1.69 HMDB0000742 C4H9NO2S Phenacemide 179.0805 8.17 HMDB0015253 C9H10N2O2 Dihydroactinidiolide 181.1218 9.56 HMDB0035240 C11H16O2 2-Aminobenzoic acid 138.0545 5.86 HMDB0001123 C7H7NO2 Hippuric acid 180.0649 5.87 HMDB0000714 C9H9NO3 1-(beta-D-Ribofuranosyl)-1,4-dihydronicotinamide 257.1149 3.9 HMDB0011648 C11H16N2O5 2-Deoxygalactopyranose 165.0708 1.54 HMDB0012327 C6H12O5 D-Fructofuranose 1,2':2,3'-dianhydride 325.1125 1.57 KEGG C04420 C12H20O10 Trehalose 343.1241 1.58 HMDB0000975 C12H22O11 4-Trimethylammoniobutanoic acid 146.1172 4.89 HMDB0001161 C7H15NO2 Glycerophosphocholine 258.1094 1.36 HMDB0000086 C8H20NO6P LysoPC(14:0) 468.308 10.33 HMDB0010379 C22H46NO7P LysoPC(16:0) 497.3435 11.69 HMDB0010382 C24H50NO7P LysoPC(P-16:0/0:0) 480.3433 12.11 HMDB0010407 C24H50NO6P PC (16:0) 482.3585 12.14 METLIN40386 C24H52NO6P LysoPE(16:0) 454.2916 11.62 HMDB0011473 C21H44NO7P LysoPE(18:1) 480.3054 11.95 HMDB0011505 C23H46NO7P LysoPE(18:2) 478.2915 11.07 HMDB0011507 C23H44NO7P SAH 385.1299 3.31 HMDB0000939 C14H20N6O5S 5'-Methylthioadenosine 298.0969 4.77 HMDB0001173 C11H15N5O3S SAM 399.1452 1.36 HMDB0001185 C15H22N6O5S 2-Acetylpyrazine 123.055 2.14 HMDB0031861 C6H6N2O L-Kynurenine 209.0919 4.34 HMDB0000684 C10H12N2O3 2-Piperidinone 100.0754 4.44 HMDB0011749 C5H9NO Lumichrome 243.0882 7.08 KEGG C01727 C12H10N4O2 1-Methyladenosine 282.1196 2.29 HMDB0003331 C11H15N5O4 Adenosine 268.1043 3.69 HMDB0000050 C10H13N5O4 N2,N2-Dimethylguanosine 312.1289 4.39 HMDB0004824 C12H17N5O5 Succinyladenosine 384.1149 4.37 HMDB0000912 C14H17N5O8 Guanosine monophosphate 364.0642 2.13 HMDB0001397 C10H14N5O8P 1-Methylguanine 166.0719 2.38 HMDB0003282 C6H7N5O Allopurinol 137.0447 3.82 HMDB0014581 C5H4N4O Guanine 152.0564 3.77 HMDB0000132 C5H5N5O Hypoxanthine 137.0445 2.42 HMDB00157 C5H4N4O 1-methylnicotinamide 173.0478 1.09 PubChem 1005-24-9 C7H9ClN2O 2-Methylnicotinamide 137.0703 8.17 HMDB0059711 C7H8N2O N1-Methyl-2-pyridone-5-carboxamide 153.0653 3.88 HMDB0004193 C7H8N2O2 Niacinamide 123.0553 2.31 HMDB0001406 C6H6N2O Cytidine 244.0907 1.92 HMDB0000089 C9H13N3O5 CDP-ethanolamine 447.0676 1.42 HMDB0001564 C11H20N4O11P2 Cytosine 112.0497 1.91 HMDB0000630 C4H5N3O Thymine 127.0498 4 HMDB0000262 C5H6N2O2 Uracil 113.0338 3.4 HMDB0000300 C4H4N2O2 Pyrrolidine 72.0806 1.94 HMDB0031641 C4H9N Acetylcholine 146.1174 1.5 HMDB0000895 C7H15NO2 Choline 104.1066 1.32 HMDB00097 C5H13NO L-Carnitine 162.1111 1.38 HMDB0000062 C7H15NO3 Phosphorylcholine 184.0724 1.34 HMDB0001565 C5H14NO4P 3-methyl-2-Quinoxalinone 161.0717 8.16 PubChem 26384 C9H8N2O METABOLITES_END #END