#METABOLOMICS WORKBENCH spinelli_lab_20251112_115953 DATATRACK_ID:6670 STUDY_ID:ST004392 ANALYSIS_ID:AN007339 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 POSITIVE MS:MS_COMMENTS The mass spectrometer was set to full scan (70-1000 m/z), with the spray voltage MS:MS_COMMENTS set to 4.0 kV, heated capillary to 350°C, and the HESI probe at 30 °C. The MS:MS_COMMENTS sheath gas flow was set at 10 units, auxiliary gas at 1 units, and sweep gas MS:MS_COMMENTS flow at 1 unit. The resolution of scan was set to 70,000, AGC target to 1x106, MS:MS_COMMENTS and maximum injection time at 20 msec. Data acquired by Thermo Fisher's Xcalibur MS:MS_COMMENTS software and analyzed by their Tracefinder software. The raw files provided MS:MS_COMMENTS 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 Anserine 48476536.12 75449727.8 69813857.09 40911700.95 62292381.66 52441958.25 35078924.16 44719927.69 85325218.19 69787389.13 50235757.81 41567956.94 94781927.48 31137545.63 34012667.26 35151033.84 Arginine 1205366794 1540542057 1651999353 1178466465 1742346414 1557307906 1404605575 1316565551 2594492864 1739562665 1428919482 1163042359 2962335080 1109520561 1055406928 1184668475 Asparagine 16976163.54 19255772.8 21075566.68 16100276.97 20178518.05 21554148.13 16658958.7 18331892.55 29839499.62 17209764.53 12785002.72 12037811.06 34174540.85 15575927.95 11066432.99 12018178.41 NAD+ 79150131.46 116391489.1 103486663.6 81506390.02 106620921.5 94626509.88 76238203.68 87841457.3 112983458.9 75491902.29 53008856.19 40276690.06 82456300.44 63637354.3 38339550.93 49204476.5 Carnosine 129259094.2 164070452.7 140958264.7 116606182.6 118724336.8 105099985.2 77669870.29 105175186.3 105141764.1 111326896.1 97041908.54 84325915.2 128485432.9 63176453.08 110991313.1 71229768.36 Choline 273740531.7 258726883.6 333283309.7 201109612.5 393440176.4 314193888.9 237182628.5 247143646.8 564304930 250657075.4 211774823 228403227.9 509420429.4 151789625.1 143188184.3 207198133.4 Creatine 52663651383 56078792349 55468171250 51496667621 57083017306 53598944876 52333261346 52897116667 65726360175 60749459740 55270941537 51679447558 68717281905 51818247340 50564343156 50531929615 Cystathionine 20175118.76 29082947.87 29290066.76 17800850.34 25312693.62 23710343.84 20303423.22 18839584.08 66755970.02 41584571.66 32480618.83 29838543.47 70549131.6 22612159.91 22193303.44 26677497.16 Cystine 255900.3127 403530.3793 207662.0352 0 177627.8542 835793.4104 2238654.392 1846834.875 78185.63587 99999.05862 157625.9191 287057.0154 35117.54184 97070.4607 298999.7655 0 Cytidine 164031837.5 186069267.9 202205307 107489145.9 249013014.7 177005027 117902650.7 147399688.3 428685901 202526900.4 171023170.6 138273421.1 448955412.7 114809598.1 98529514.32 123699018.7 Cytosine 553564916.4 608815265.6 669542544.5 358745719.7 846090049.7 603353362.4 401239315.9 506871504.7 1423114163 671436772.5 583181931.7 472015535 1484155667 410860464.6 331080828 435239098 Alanine 1152666269 1221911017 1191634062 1132539630 1261108468 1196138613 1138047229 1153037543 1453322102 1328739167 1223006650 1136834087 1498228915 1138244827 1114773199 1109010746 D-Ornithine 12627432.23 14723756.68 15872142.83 9186599.331 18503573.92 14119088.1 14914598.05 12971857.46 21682259.19 15560306.58 16075218.76 13281043.5 20580062.08 12584798.14 9277349.291 12823053.98 FAD 6862167.137 9271569.459 8201244.83 6202547.016 8297459.616 7904294.935 6173081.919 5720774.248 17657880.72 11254355.79 7110901.653 4948238.99 24032330.37 5172769.818 5862888.889 5640955.79 Glutamine 2823961954 3070084229 3103853384 2940568299 2707505899 2859234426 2963661423 2473257464 3873757452 3220769881 2870067964 2665006372 3770372400 2825516847 4302629156 2748549662 Glutathione 1218955194 1386190108 1524965166 1337197242 1526434159 1398795885 1074736009 1138289810 2034737693 1608134766 1365443149 1022216793 2572892237 1185082288 1133487754 1137113453 Glycine 3529023.471 4121069.455 4566576.349 3472148.201 4700382.218 3675106.738 4866176.156 5021378.305 6230131.635 3992111.707 3551595.716 3365945.517 6681888.85 3290151.511 3045963.969 3964701.668 Oxidized glutathione 135620562.2 189085759.3 195963869.1 96579794.57 181209151.2 136337079.8 93938762.21 102161821.4 342945427.6 192942344.2 108825151.2 103065485.1 429969263.3 84602071.51 79706320.53 102172687.1 Guanine 15905055.4 24902285.63 16192295.51 14870166.28 15996159.79 14424232.67 14058272.16 12700250.46 28638692.07 25268256.26 18508994.85 10525168.72 36199579.57 11658724.36 10029656.31 10377797.79 Homocysteine 1765487.423 2870105.74 2904033.36 1935378.921 2518609.654 3021230.191 2314252.909 1811510.681 4817912.191 3376628.277 2350817.502 2025641.969 6367954.24 2045773.047 1968285.113 2231437.336 Homoserine 64968372.39 61195042.73 58652024.14 62870432.22 66450550.05 62357823.9 67464089.96 49192235.11 72027947.71 56232599.33 61511848.45 55022785.78 82071518.34 60088765.44 37882282.62 67674450.89 Hypotaurine 10590876.75 11375784.21 14316340.45 9578336.007 13412280 12653963.94 11195788.91 12740335.37 34762604.59 23109465.82 16021095.79 15328584.68 50955171.76 17323932.77 4874641.117 18020114.35 Hypoxanthine 3606492760 2479095899 3750031990 2115413781 4532478188 3254434858 2472070877 2818699549 8044822002 3572369377 3733368679 4265194750 4981403954 2784737218 2382448232 3020869471 Inosine 386185398.3 287471360.7 429923184.6 209474780.2 544674634.8 426115478.3 421962470.4 339764171 898087905.3 374108848.4 417332738.3 416891801.3 611898701.4 336361152.7 228401624.9 318221360.4 Isoleucine 107418233.9 129916021.9 161862168 96060497.52 172013379.4 154299125.7 126944268.3 118446814.4 315597020.3 146909031.4 116540272.5 112313351.5 375161737.9 117938519 97970619.82 110455696.9 Kynurenine 349508.0711 761550.2289 1729697.825 638373.6813 775754.9779 791776.1919 595621.3401 413083.5469 3205702.837 1684976.087 860744.0126 461298.2634 3713320.46 884025.2123 829727.0366 621382.9273 Carnitine 7327441553 9861003511 10338518688 7492753893 10862300699 11067411571 9274976810 8204211348 19433597843 11741680364 8875755857 7870152259 23571476737 7928639572 6865219413 8712823374 Leucine 166512156.7 224676651.3 255363292.3 165747939.2 252873648.2 232441865.2 197717932.6 178970650.8 504451637.6 255447288.2 191956235.3 165153457.4 492151589 176894099.5 168956917.9 157685623.4 Lysine 304597933.1 435852321.7 499631126.4 281022265.9 549128057 475838102.9 417613405.9 399444709.4 917543230.2 523838620.8 370978827.9 319312418.8 1194535425 328884334.6 257743806.2 346052546.7 Methionine 340142795.9 543815454.8 341091720.5 341137008.3 311310627.9 304576252.5 263352938.5 248885731.9 593869352.6 569015193 411557093.1 228469008.8 757854203.2 241322268.8 208665271.1 222183241.6 NADP+ 5348078.888 7398470.173 7689053.169 5979938.176 6972483.563 5230378.98 4982036.743 5871679.739 8407925.75 4711018.295 3209430.169 1974515.308 8820010.151 3884138.362 2909545.342 3178539.989 NADPH 104455.3959 0 50127.49158 577991.1395 0 221036.829 0 0 5084863.936 3836503.017 1689122.971 1076030.552 7302616.948 576163.3771 1249168.178 1283031.593 Niacinamide 8615058120 9172787743 9782141469 6769983142 10325303979 9613260962 8719525012 9161068406 14710000145 12293701213 10906561222 10405478559 16699095892 8166420364 10156347536 9581325017 Phenylalanine 406819181.6 568982861.6 685946985.6 395701291.1 666852360.7 590065226.8 490901940.5 398786027.5 1376308063 734097964.7 486155237.9 409847372.1 1414296239 401862665.6 577467751.2 403344668.9 Proline 1978060745 2419749201 2625003907 1643416297 2755895781 2552528556 2104676728 1875705170 4351558910 2593987173 1991167868 1691541165 4931074589 1739709936 1509286595 1787180627 S-Adenosylhomocysteine 44898588.91 51938654.7 57634673.1 30872913.55 70667470.27 54922451.11 38297331.3 37735020.62 94611954.42 47528292.44 39016854.69 31755821.36 81489756.64 24894713.54 28922373.87 30582850.62 S-Adenosylmethionine 161443407.4 181529042.8 202848515.2 156648152.6 184201346.6 184525364.8 164746576.2 171210997.5 190284214.6 180285275.7 159078945.4 152994641.6 232796018.3 166400484.3 138665152.9 160270343.2 Serine 41744784.28 48424758.59 51978772.97 39959593.19 53677495.31 48810647.88 43772199.43 43303580.94 79345601.54 51276439.6 42330483.98 36003899.13 91334372.72 38705059.34 34150407.46 38083235.7 Taurine 2392957916 3048096731 3190526693 2404208839 3313730948 2905144182 2515788166 2659436000 3960024937 3009445906 2279519539 1944310715 5145929945 2110140005 1409137427 2034749856 Threonine 64769858.35 61158915.53 58626118.36 62381826.2 65684630.64 62039004.15 67452886.58 48937269.73 71985985.37 56197555.31 61511848.45 55022785.78 81950186.28 60088765.44 37874425.29 67669496.26 Tryptophan 67033373.52 111596175.8 114558410.8 76520826.15 109874278.4 112533544.5 95024421.89 85930585.05 223088022.1 128845895.6 84842842.72 74537349.41 316662765.9 85964513.13 134828138.3 83254707.71 Tyrosine 200293783.9 263630478 313141663.3 159335758.7 227473266 271749829.2 219483100.4 167841821.6 386908915.9 301733697.1 222269239 181318127 460917543.9 206743027.7 247406868.4 191169617 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity Anserine 7.4 241.1295 112072 C01262 Positive Arginine 13 175.119 6322 C00062 Positive Asparagine 8.26 133.0608 6267 C00152 Positive NAD+ 8 664.1164 5893 C00003 Positive Carnosine 8.4 227.1139 439224 C00386 Positive Choline 9.75 105.1148 305 C00114 Positive Creatine 8 132.0768 586 C00300 Positive Cystathionine 9.5 223.0747 439258 C02291 Positive Cystine 9.3 241.0311 67678 C00491 Positive Cytidine 6.13 244.0928 6253 C00475 Positive Cytosine 6 112.0505 597 C00380 Positive Alanine 7.9 90.055 5950 C00041 Positive D-Ornithine 11.5 133.0972 71082 C00515 Positive FAD 6.3 786.1644 643975 C00016 Positive Glutamine 8.1 147.0764 5961 C00064 Positive Glutathione 8.5 308.0911 124886 C00051 Positive Glycine 8.5 76.0393 750 C00037 Positive Oxidized glutathione 10.5 613.1592 65359 C00127 Positive Guanine 5.7 152.0567 135398634 C00242 Positive Homocysteine 9.38 136.0427 91552 C00155 Positive Homoserine 7.7 120.0655 12647 C00263 Positive Hypotaurine 8.15 110.027 107812 C00519 Positive Hypoxanthine 5.21 137.0458 135398638 C00262 Positive Inosine 5.59 269.0881 135398641 C00294 Positive Isoleucine 5.5 132.1019 6306 C00407 Positive Kynurenine 5.4 209.0921 161166 C00328 Positive Carnitine 7 162.1125 10917 C00318 Positive Leucine 5.3 132.1019 6106 C00123 Positive Lysine 12.6 147.1128 5962 C00047 Positive Methionine 5.7 150.0583 6137 C00073 Positive NADP+ 10 744.0827 5886 C00006 Positive NADPH 10 746.0984 5884 C00005 Positive Niacinamide 3.33 123.0553 936 C00153 Positive Phenylalanine 4.87 166.0863 6140 C00079 Positive Proline 6.76 116.0706 145742 C00148 Positive S-Adenosylhomocysteine 7 385.1289 439155 C00021 Positive S-Adenosylmethionine 8.7 400.1523 34756 C00019 Positive Serine 8.5 106.0499 5951 C00716 Positive Taurine 8.1 126.0219 1123 C00245 Positive Threonine 7.8 120.0655 6288 C00188 Positive Tryptophan 5.9 205.0972 6305 C00078 Positive Tyrosine 6.65 182.0812 6057 C00082 Positive METABOLITES_END #END