#METABOLOMICS WORKBENCH spinelli_lab_20251112_115953 DATATRACK_ID:6670 STUDY_ID:ST004392 ANALYSIS_ID:AN007340 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 20, 2025, 4:51 pm #PROJECT PR:PROJECT_TITLE Metabolic Reprogramming during Human Neuron Differentiation Indicates PR:PROJECT_TITLE Glutaminase as a Key Determinant in Fragile X Syndrome PR:PROJECT_SUMMARY Metabolic homeostasis gone awry is a contributor to, if not an underlying cause PR:PROJECT_SUMMARY of, several neurologic disorders. Fragile X syndrome (FXS) is a PR:PROJECT_SUMMARY neurodevelopmental disorder caused by a trinucleotide repeat expansion in FMR1 PR:PROJECT_SUMMARY and consequent loss of the encoded protein FMRP, which results in downstream PR:PROJECT_SUMMARY molecular, neurologic, and mitochondrial deficits that are linked to cognitive PR:PROJECT_SUMMARY impairment. In human postmortem brain, many metabolites and solute carrier PR:PROJECT_SUMMARY proteins are coordinately dysregulated, which also occurs during differentiation PR:PROJECT_SUMMARY of human iPSCs into excitatory neurons. Metabolic tracing in FXS neurons PR:PROJECT_SUMMARY demonstrates a dearth of glutamine deamidation to glutamate, which reduces PR:PROJECT_SUMMARY anaplerosis into the TCA cycle, potentially hindering bioenergetic and PR:PROJECT_SUMMARY biosynthetic functions of mitochondria. Mechanistically, aberrant expression of PR:PROJECT_SUMMARY glutaminase isoforms in FXS is responsible for reduced glutaminolysis, thereby PR:PROJECT_SUMMARY altering glutamate levels which may contribute to FXS. PR:INSTITUTE UMass Chan Medical School PR:LAST_NAME UMass Chan PR:FIRST_NAME Spinelli Lab PR:ADDRESS 55 Lake Avenue North, Worcester, Massachusetts, 01605, USA PR:EMAIL spinellilab@gmail.com PR:PHONE (508) 856-8989 ext. 68148 #STUDY ST:STUDY_TITLE Polar metabolic differences in cortex and hippocampus of wild-type (WT) and Fmr1 ST:STUDY_TITLE KO mice. ST:STUDY_SUMMARY Polar metabolites profiling in cortex or hippocampus of WT versus Fmr1 KO mice. ST:INSTITUTE UMass Chan Medical School ST:LAST_NAME UMass Chan ST:FIRST_NAME Spinelli Lab ST:ADDRESS 55 Lake Avenue North, Worcester, Massachusetts, 01605, USA ST:EMAIL spinellilab@gmail.com ST:PHONE (508) 856-8989 ext. 68148 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Male #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - WT1 hpc Sample source:Male WT Mouse Hippocampus | Genotype:WT RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_01.raw SUBJECT_SAMPLE_FACTORS - WT2 hpc Sample source:Male WT Mouse Hippocampus | Genotype:WT RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_02.raw SUBJECT_SAMPLE_FACTORS - WT3 hpc Sample source:Male WT Mouse Hippocampus | Genotype:WT RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_03.raw SUBJECT_SAMPLE_FACTORS - WT4 hpc Sample source:Male WT Mouse Hippocampus | Genotype:WT RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_04.raw SUBJECT_SAMPLE_FACTORS - KO1 hpc Sample source:Male Fmr1 KO Mouse Hippocampus | Genotype:Fmr1 KO RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_05.raw SUBJECT_SAMPLE_FACTORS - KO2 hpc Sample source:Male Fmr1 KO Mouse Hippocampus | Genotype:Fmr1 KO RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_06.raw SUBJECT_SAMPLE_FACTORS - KO3 hpc Sample source:Male Fmr1 KO Mouse Hippocampus | Genotype:Fmr1 KO RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_07.raw SUBJECT_SAMPLE_FACTORS - KO4 hpc Sample source:Male Fmr1 KO Mouse Hippocampus | Genotype:Fmr1 KO RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_08.raw SUBJECT_SAMPLE_FACTORS - WT1 ctx Sample source:Male WT Mouse Cortex | Genotype:WT RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_09.raw SUBJECT_SAMPLE_FACTORS - WT2 ctx Sample source:Male WT Mouse Cortex | Genotype:WT RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_10.raw SUBJECT_SAMPLE_FACTORS - WT3 ctx Sample source:Male WT Mouse Cortex | Genotype:WT RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_11.raw SUBJECT_SAMPLE_FACTORS - WT4 ctx Sample source:Male WT Mouse Cortex | Genotype:WT RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_12.raw SUBJECT_SAMPLE_FACTORS - KO1 ctx Sample source:Male Fmr1 KO Mouse Cortex | Genotype:Fmr1 KO RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_13.raw SUBJECT_SAMPLE_FACTORS - KO2 ctx Sample source:Male Fmr1 KO Mouse Cortex | Genotype:Fmr1 KO RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_14.raw SUBJECT_SAMPLE_FACTORS - KO3 ctx Sample source:Male Fmr1 KO Mouse Cortex | Genotype:Fmr1 KO RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_15.raw SUBJECT_SAMPLE_FACTORS - KO4 ctx Sample source:Male Fmr1 KO Mouse Cortex | Genotype:Fmr1 KO RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_16.raw #COLLECTION CO:COLLECTION_SUMMARY Wild-type and Fmr1 KO mice used were purchased from Jackson laboratories and CO:COLLECTION_SUMMARY were housed under a 12 h light/dark cycle with routine monitoring and with free CO:COLLECTION_SUMMARY access to food and water. 3-month-old male mice were used in this study (n = 4 CO:COLLECTION_SUMMARY each for WT and Fmr1 KO). CO:SAMPLE_TYPE Mouse Brain Hippocampus and Cortex #TREATMENT TR:TREATMENT_SUMMARY Wild-type and Fmr1 KO mice used were purchased from Jackson laboratories and TR:TREATMENT_SUMMARY were housed under a 12 h light/dark cycle with routine monitoring and with free TR:TREATMENT_SUMMARY access to food and water. 3-month-old male mice were used in this study (n = 4 TR:TREATMENT_SUMMARY each for WT and Fmr1 KO). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Tissues were powdered under liquid nitrogen and samples were normalized based on SP:SAMPLEPREP_SUMMARY tissue weight. The metabolites were extracted using 80% methanol. The extracts SP:SAMPLEPREP_SUMMARY were spun down and the supernatant was vacuum dried before resuspending in LC/MS SP:SAMPLEPREP_SUMMARY grade water. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) CH:SOLVENT_A 100% water; 0.1% ammonium hydroxide; 20mM ammonium carbonate CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 20 min, 80% - 20% B; 0.5 min, 20% - 80% B; 7.5min, 80% B CH:FLOW_RATE 0.15ml/min CH:COLUMN_TEMPERATURE 25 #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 NEGATIVE MS:MS_COMMENTS The mass spectrometer had the spray voltage set to 4.0 kV, heated capillary to MS:MS_COMMENTS 350°C, and the HESI probe at 30 °C. The sheath gas flow was set at 10 units, MS:MS_COMMENTS auxiliary gas at 1 units, and sweep gas flow at 1 unit. An additional scan MS:MS_COMMENTS between 220-700 m/z was used to enhance nucleotide detection in the negative MS:MS_COMMENTS mode as well with the maximum injection time set to 80 msec. Data acquired by MS:MS_COMMENTS Thermo Fisher's Xcalibur software and analyzed by their Tracefinder software. MS:MS_COMMENTS The raw files provided contain data from both positive and negative ion mode. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples WT1 ctx WT2 ctx WT3 ctx WT4 ctx KO1 ctx KO2 ctx KO3 ctx KO4 ctx WT1 hpc WT2 hpc WT3 hpc WT4 hpc KO1 hpc KO2 hpc KO3 hpc KO4 hpc Factors Sample source:Male WT Mouse Cortex | Genotype:WT Sample source:Male WT Mouse Cortex | Genotype:WT Sample source:Male WT Mouse Cortex | Genotype:WT Sample source:Male WT Mouse Cortex | Genotype:WT Sample source:Male Fmr1 KO Mouse Cortex | Genotype:Fmr1 KO Sample source:Male Fmr1 KO Mouse Cortex | Genotype:Fmr1 KO Sample source:Male Fmr1 KO Mouse Cortex | Genotype:Fmr1 KO Sample source:Male Fmr1 KO Mouse Cortex | Genotype:Fmr1 KO Sample source:Male WT Mouse Hippocampus | Genotype:WT Sample source:Male WT Mouse Hippocampus | Genotype:WT Sample source:Male WT Mouse Hippocampus | Genotype:WT Sample source:Male WT Mouse Hippocampus | Genotype:WT Sample source:Male Fmr1 KO Mouse Hippocampus | Genotype:Fmr1 KO Sample source:Male Fmr1 KO Mouse Hippocampus | Genotype:Fmr1 KO Sample source:Male Fmr1 KO Mouse Hippocampus | Genotype:Fmr1 KO Sample source:Male Fmr1 KO Mouse Hippocampus | Genotype:Fmr1 KO 2-Phosphoglyceric acid 31661570.2 99061905.79 63296809.4 50658059.37 41656531.92 33888763.17 62910426.79 31651862.7 222924663.5 165938914.4 93452310.65 37832142.29 215331061.4 58619950.34 55731167.39 44894633.2 3-Phosphoglyceric acid 31661570.2 99061905.79 63296809.4 50658059.37 41656531.92 33888763.17 62910426.79 31651862.7 222924663.5 165938914.4 93452310.65 37832142.29 215331061.4 58619950.34 55731167.39 44894633.2 Adenosine 14673353.18 17632504.72 16406890.68 11709734.76 19013588.51 15596517.19 14173252.27 19058306.17 13585518.32 8516412.318 6874965.248 6997669.672 25413287.51 6690788.004 12888181.88 7436291.521 ADP 265208000.8 478289220.6 434809721.1 248676188.9 373029459 368304194.5 276062749.8 308069342.3 589156274.5 446983377.4 264070888.4 186115800 810277217.2 225039127 234046391.3 216668021.1 ATP 56352286.37 118096993.7 108223706.2 86368192.63 93682817.32 93676781.58 62289964.12 74386604.23 139330377.2 130841326.1 79062137.79 44679816.55 179609672.3 55310549.45 77203559.46 61940614.33 AMP 1408272935 2064458447 2006635287 1476686666 1887465886 1692536964 1454492621 1488794825 2922676151 2230807599 1409909800 1115732375 4445793731 1254144445 1213696981 1199714025 Azelaic acid 2749817.716 3910975.35 3213028.009 2139054.663 2773485.058 1818312.135 1686644.575 1767648.361 4590481.397 4315507.189 2520902.462 1590150.727 5958144.14 1159986.66 1326542.738 1489076.063 NADH 61601227.09 106068864.3 127838000 105855167.9 110086591.1 110882406.3 120004123.5 71982665.45 76905914.46 38121338.9 20763767.97 10181403.33 114488685.5 20744119.52 14662678.26 26513940.42 Citric acid 436835216.6 571440357.8 624108701 406561352.8 621059184.2 507959268.2 494794197.6 493135844.6 741627699.1 606141956.7 437173863 353649177.4 1047213937 303819684.8 355868770.7 390731042.7 3',5' cyclic AMP 2401310.406 2672013.008 2770191.432 2177036.157 3680104.744 2012241.567 1092895.095 2293037.638 11459592.85 6791014.117 4068645.033 2950311.73 19131801.79 3729784.26 4034094.141 3733862.267 CDP 2236334.141 5950685.087 4957495.457 2623627.051 3145847.046 2859667.648 3202687.818 4018230.612 3864069.485 3479397.454 1768425.943 1250401.542 7200845.715 1316055.805 1327673.513 1591723.272 CMP 15630459.58 30368907.46 26211218.94 22196761.59 19006730.27 15994362.6 19145722.95 19957530.43 36560752.17 38598973.38 19162203.1 14935879.58 81164847.29 14128096.7 13372405.46 16683726.24 CTP 291641.6834 1313918.082 1142200.237 604927.5296 694397.2805 665565.8084 610147.9806 678783.201 532745.2285 620686.6406 269767.2847 105540.8168 948665.0395 186245.51 154216.4115 217177.7886 beta-D-sedoheptulose 114300.777 71734.43027 42863.09737 77929.32675 103500.5411 152357.4428 158400.6599 46587.75106 290031.3152 184702.6131 148209.2523 115304.209 221643.8748 206906.4392 207097.3281 131535.0002 Fumaric acid 57310631.84 62726029.38 70587985.26 51699048.44 76200460.16 58546208.53 55012372.17 57188328.04 106754775.9 74054746.61 61517510.39 54030374.24 147797231.2 47579091 44412791.29 56009470.12 Galactose 138428978.4 190496201 187663141.6 125031631.9 167772063.7 147730179 119194323.9 126916308.6 441705321.1 242275486.3 177083823.5 123164282.5 544375566.6 106681799.5 148906222.8 117444176.5 Gluconic acid 27979122.44 34242372.47 37629801.77 22958463.22 43734309.06 39210919.38 30798655.85 31487270.5 52741360.57 31043608.34 25195601.13 22187628.85 63877428.27 24118126.43 21496431.62 22822539.56 Glucosamine 6-phosphate 76899555.06 107457224.6 102333674.6 87450631.11 89314073.94 64522552.67 95162640.33 91383539.55 104574450.1 128965241.3 94835913.71 73531312.3 254134261.8 81912777.42 103763195.3 66384166.94 Glutamic acid 867575987.8 1192115285 1131429981 825069923.1 1062637923 921718732.2 813420218.1 823991848.7 1573126020 1227768645 904896480.8 698612892.3 2075923755 695491155.8 790658204.2 725040721.4 Glycerol 1980764.843 2011179.954 2396942.044 1205074.263 2820216.26 2652764.362 1542712.584 1278571.118 6006694.575 2409841.835 1995518.6 1505183.645 7231351.417 1471536.099 1635448.459 1435352.663 sn-Glycero-3-phosphate 35961247.67 45600167.19 37508897.53 25020800.3 36848427.43 33916459.21 26200033.27 17113863.35 76822104.82 41160102 33413661.24 42041.15277 44884011.56 28557575.41 19426406.2 16496536.61 Guanosine 54468446.1 45720555.45 59715042.14 27989490.66 85690551.63 72511340.29 74259059.15 66184583.45 116339595.8 33349029.76 39011584.43 40440010.97 95494653.52 35819646.64 26533362.8 34499694.92 Isocitric acid 436109090.8 569955251.3 623165706.9 444938277.8 620646715.7 507283933.9 494168818.1 492027353.2 744849846.1 605626210.5 436384127.3 352193569.2 1045454838 303722823 355509451.5 390512525.6 Itaconic acid 16173421.86 19427010.99 19683037.31 16265610.41 16825020.49 14336173.78 13413341.23 14315594.48 26752272.51 16567998.52 13073840.03 11845446.39 30976496.23 11430937.85 15409873.51 11956207.71 Lactate 1408856951 1909003820 2157776723 1212946244 2268231363 1896582300 1454572733 1428456322 4654556036 2081705340 1467989437 1261093095 6268215289 1361208790 1228917716 1270441796 Malic acid 417363615.9 457697173.6 510750901.1 390736027.4 559274302 429445846.6 383357269.1 421575461.4 752956877.9 539522504.7 470121557.9 408668169.7 1058939072 358884206.4 337218770.3 415250925.7 Mannitol 9678859.096 11070682.73 12183992.94 7463081.396 11418033.29 10434850.3 7801496.813 8480923.895 17892944.09 11941251.73 9001001.978 7740141.573 23776948.58 8769339.603 5876354.318 7216784.568 Methylmalonic acid 56339286.37 89196465.44 81910099.18 54581105.84 83293460.53 91534667.2 56033200.72 50283983.71 195776910.1 84061177.12 57911652.52 39076910.94 151714542.4 52455027.24 52096601.05 38356643.55 N-Acetylaspartic acid 5695360538 7009975120 7022406527 5422189517 6885737332 6388187357 5653548477 5780714405 8248430771 6403699432 5094254070 4350967104 9994150145 4514757715 4519950525 4509579386 Orotic acid 2937348.523 4730484.316 4355512.17 3103875.414 3776395.122 3862435.316 3321106.051 3154458.297 4120180.486 2429058.058 2022571.841 1699393.369 5291258.927 1823799.499 2131345.461 1944848.53 Phosphocreatine 1303001.003 2535521.031 2022793.583 90892.50927 1949275.896 1650157.301 2064676.33 2022445.889 1432709.848 607702.4539 390858.9055 389362.7924 3508203.549 540118.2549 488541.8232 363235.6826 Phosphoenolpyruvic acid 9326788.461 24459659.88 18922362.39 9915660.579 11094902.98 15871692.04 24217697.72 10799512.71 53877271.6 36857035.38 19058705.54 9954425.123 50723281.83 11937478.08 13967590.68 9681028.228 Pyruvic acid 6089990.331 10915333.21 14983257.97 8512175.213 12320839.03 14479945.73 11363522.96 12963030.14 10262809.59 8214392.722 4271834.928 3412998.774 22309457.61 4717737.771 4766653.758 5362871.304 Succinic acid 63044096.8 100407543.8 90039779.13 59418853.55 91448443.91 102649911.8 63370623.97 56396069.76 220463847.7 92567072.55 65490945.44 42299683.21 174171041 58980817.79 57295888.49 41962216.9 Uracil 11360203.22 10556586.54 13602098.23 4253046.727 20114874.04 17436349.59 7325504.89 8219304.207 25292420.74 9533534.62 10248502.19 9406941.51 17349123.46 6185802.457 5269215.352 11530371.53 Uric acid 3451625.026 3352304.648 5166168.816 2886599.912 6244359.553 5908812.135 4630360.481 4589745.628 4755717.541 3013912.381 2568405.294 4494794.186 4138833.084 4526972.647 7978814.145 4520646.348 Uridine 43666407.69 36081990.05 50186556.14 26109198.91 72344623.55 54912026.5 35618996.17 38440486.11 83648838.21 34808304.65 37931506.84 32893968.32 82054298.43 34162993.34 27585170.1 34230427.32 UDP 59014247.96 132930073.5 117488267.5 61256744.76 82994248.64 75126250.75 74337101.44 87487782.25 82022479.18 77379839.23 47661974.25 30739311.02 116413041.2 35088967.59 38474281.7 40534877.67 UMP 97549986.24 184963964.3 164779019.3 145866931.4 120748074.9 104517684.2 122025726.1 130997403.6 187937598.1 211085076.1 110026584 86573163.27 424351874.6 89620280.24 79413188.14 107914034.3 UTP 1915657.463 6476815.32 5435144.425 3159857.119 3945717.074 3713334.423 2865152.685 2965295.847 2908406.271 3224160.051 1672575.819 930984.4322 5295812.861 1307684.741 1352294.549 1444545.295 Xanthosine 1330875.286 1082528.758 1463555.781 752908.1902 2399983.75 2328004.961 1376845.832 1114759.544 3600704.28 1337313.43 1635417.917 1738791.233 2872800.632 1531661.749 1665426.788 1560756.092 Xylose 1045182.629 1453584.671 1501563.419 853577.2381 1351132.713 1116869.083 865014.1872 970001.1367 3108653.117 2867755.443 1146469.442 898756.3151 7389540.474 1048283.282 1046378.134 973481.2295 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity 2-Phosphoglyceric acid 10.2 184.9857 59 C00631 Negative 3-Phosphoglyceric acid 10 184.9857 724 C00597 Negative Adenosine 4.4 266.0895 60961 C00212 Negative ADP 8.9 426.0221 6022 C00008 Negative ATP 10 505.9885 5957 C00002 Negative AMP 8 346.0558 6083 C00020 Negative Azelaic acid 6.2 187.0976 2266 C08261 Negative NADH 7.7 664.1175 439153 C00004 Negative Citric acid 10.9 191.0197 311 C00158 Negative 3',5' cyclic AMP 5 328.0452 6076 C00575 Negative CDP 10.2 402.0109 6132 C00112 Negative CMP 9.4 322.0446 6131 C00055 Negative CTP 11.3 481.9772 6176 C00063 Negative beta-D-sedoheptulose 7.5 209.0667 441483 C08355 Negative Fumaric acid 9.5 115.0037 444972 C00122 Negative Galactose 9.69 179.0561 439357 C00984 Negative Gluconic acid 7.8 195.051 10690 C00257 Negative Glucosamine 6-phosphate 9 258.0384 439217 C00352 Negative Glutamic acid 8.5 146.0459 33032 C00025 Negative Glycerol 5.23 91.0401 753 C00116 Negative sn-Glycero-3-phosphate 8.65 171.0064 439162 C00093 Negative Guanosine 6.5 282.0844 135398635 C00387 Negative Isocitric acid 10.8 191.0197 1198 C00311 Negative Itaconic acid 9 129.0193 811 C00490 Negative Lactate 5.16 89.0244 107689 C00186 Negative Malic acid 9.5 133.0143 222656 C00149 Negative Mannitol 7.5 181.0718 6251 C00392 Negative Methylmalonic acid 8.8 117.0193 487 C02170 Negative N-Acetylaspartic acid 8.5 174.0408 65065 C01042 Negative Orotic acid 5.8 155.0098 967 C00295 Negative Phosphocreatine 9 210.0285 587 C02305 Negative Phosphoenolpyruvic acid 10.6 166.9751 1005 C00074 Negative Pyruvic acid 4.2 87.0088 1060 C00022 Negative Succinic acid 8.8 117.0193 1110 C00042 Negative Uracil 4.7 111.02 1174 C00106 Negative Uric acid 7.1 167.0211 1175 C00366 Negative Uridine 4.8 243.0623 6029 C00299 Negative UDP 10 402.9949 6031 C00015 Negative UMP 9 323.0286 6030 C00105 Negative UTP 11 482.9613 6133 C00075 Negative Xanthosine 6.7 283.0684 64959 C01762 Negative Xylose 6.97 149.0456 135191 C00181 Negative METABOLITES_END #END