#METABOLOMICS WORKBENCH spinelli_lab_20251112_131016 DATATRACK_ID:6673 STUDY_ID:ST004394 ANALYSIS_ID:AN007344 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 20, 2025, 5:21 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 Metabolomics on Lymphoblastoid cell lines (LCL) betweeen individuals with ST:STUDY_TITLE Fragile X Syndrome and from typically developing male controls. ST:STUDY_SUMMARY Extract and measure polar metabolites from Lymphoblastoid cell lines (LCL) from ST:STUDY_SUMMARY individuals with Fragile X Syndrome versus from typically developing male ST:STUDY_SUMMARY controls. 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 Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - FXS1_01 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS101.raw SUBJECT_SAMPLE_FACTORS - FXS1_02 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS102.raw SUBJECT_SAMPLE_FACTORS - FXS1_03 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS103.raw SUBJECT_SAMPLE_FACTORS - FXS2_01 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS201.raw SUBJECT_SAMPLE_FACTORS - FXS2_02 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS202.raw SUBJECT_SAMPLE_FACTORS - FXS2_03 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS203.raw SUBJECT_SAMPLE_FACTORS - TD_01 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_WT01.raw SUBJECT_SAMPLE_FACTORS - TD_02 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_WT03.raw #COLLECTION CO:COLLECTION_SUMMARY Lymphoblastoid cell lines (LCLs) were obtained from the Coriell Institute, CO:COLLECTION_SUMMARY including three from individuals with Fragile X Syndrome (GM07365 (FXS1), CO:COLLECTION_SUMMARY GM03200 (FXS3) and GM06897 (FXS2)) and two from typically developing male CO:COLLECTION_SUMMARY controls (GM24086 (WT6) and GM06890 (WT4)). CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY The cells were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with TR:TREATMENT_SUMMARY 15% fetal bovine serum (FBS) and 2.5% L-glutamine, maintained at 37°C with 5% TR:TREATMENT_SUMMARY CO₂ in T25 flasks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After the cells were pelleted, samples were incubated on dry ice with cold 80% SP:SAMPLEPREP_SUMMARY methanol. The extracts were spun down and the supernatant was vacuum dried SP:SAMPLEPREP_SUMMARY before resuspending in LC/MS 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 FXS1_01 FXS1_02 FXS1_03 FXS2_01 FXS2_02 FXS2_03 TD_01 TD_02 Factors Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD Pyruvic acid 1479179.413 1022138.075 1300799.148 2274471.813 1671976.596 1454288.564 0 866120.2107 3-Methyl-2-oxovaleric acid 1860342.318 2558932.895 1593115.646 3201866.935 3428941.715 2757251.332 2248820.278 2494443.527 GTP 116112.8734 163995.0554 95256.3667 53643.90735 64524.94856 78698.02995 43636.62042 1616.190068 Lactate 470744861.9 641572105.7 384168934.3 368497315.2 443860026.4 262896321.3 171702826.5 191481386.6 Malic acid 64319428.39 90068809.89 91733965.4 161047980.5 145378581.7 117133435.7 102403524.1 88730159.75 GDP 970672.2432 1197997.009 892377.1363 931355.4201 940205.2123 762704.7864 699737.7501 496729.6981 Fumaric acid 4402918.575 7017488.152 6692706.68 15916299.26 12791346.86 8286351.385 9620852.526 5934617.221 Ribitol 2250440.159 2455727.623 1838786.221 3870418.9 5992629.468 2831089.813 2583978.137 2481690.665 ADP-ribose 1836333.204 2524743.484 1331229.076 2134976.033 2359857.253 1613490.37 1107180.648 1841601.411 Arabitol 2250440.159 2408644.613 0 5189464.468 5981878.892 2834312.293 2689884.108 3440294.027 Maltose 342480.3865 320673.6785 391563.7537 492728.1245 340994.9601 358884.0073 200285.0229 371481.2241 Lactose 347690.214 316916.9414 377461.6759 505994.1348 329450.9682 363429.3261 203649.062 367452.5697 Cellobiose 312704.0491 319527.4878 347834.4405 574264.5718 331544.0984 346307.868 203546.0608 369350.1274 UDP-N-acetylglucosamine 7498594.545 9817569.915 7229268.351 10270415.91 9504024.886 7380097.345 7945084.592 7076314.634 IMP 3928685.543 5493374.625 2818158.979 5790214.961 4981593.901 3574183.888 17554636.25 14187329.8 Glucosamine 6-phosphate 528885.9386 419804.6821 393657.1795 557279.013 920704.1715 557024.5723 355434.4197 651553.7031 ATP 672712.9261 811843.9318 489023.6415 381665.6196 447528.7718 462650.4469 459179.1458 238627.4061 UDP 3375719.099 4060238.724 4231366.564 4755344.464 4213515.266 2953473.139 3534231.761 3036249.656 UTP 352448.1429 371341.4237 398891.2031 356127.1372 321251.8328 324060.105 377675.0042 161267.5451 Adenosine 3',5'-diphosphate 3203212.181 3792731.192 2753674.819 3257210.83 3335683.333 2510940.057 3007026.617 2264851.38 CDP 717426.882 903429.5814 913330.6053 1068287.898 978111.8224 776380.9102 904637.4183 779893.0748 GDP-mannose 278809.5822 411521.7642 239375.3016 260528.9552 249562.4854 139343.5813 207182.9066 175389.4329 Butyric acid 62752.96745 152740.9302 90296.29036 114616.6345 0 0 98932.73632 500342.1869 Monomethyl glutaric acid 39245637.25 41394012.68 46644298.99 56591461.3 52713720.01 41594605.53 96014774 89289695.53 UDP-glucuronic acid 181054.7062 275374.1746 189103.0614 198248.1367 165268.6314 106535.4856 0 231509.7968 N-Acetylaspartic acid 6513204.352 10504789.12 5416102.188 7190403.495 5304989.834 2496261.893 15336738.08 12021357.82 Xylitol 2250440.159 2444716.224 1498591.004 4353416.944 5990384.672 2795863.149 2643194.86 5348325.829 Mannose 6-phosphate 1056107.872 1290371.395 812524.8495 1214760.571 1111895.498 985439.6977 1196184.407 1160220.49 Homovanillic acid 1726497.378 2133930.414 1447887.073 7775409.515 9655736.752 6329625.124 8736212.498 8036198.966 CDP-Ethanolamine 548015.6532 690309.849 537493.789 926444.1297 913432.7841 618345.4734 1643802.312 1371380.087 Fructose 6-phosphate 1041741.742 1303374.745 813123.6026 1253208.713 1098247.612 985439.6977 1202046.73 1212165.802 Glucose 1-phosphate 1041741.742 1303374.745 813123.6026 1253208.713 1098247.612 985439.6977 1202046.73 1212165.802 Galactose 1-phosphate 1043443.126 1292869.77 808748.4208 1214760.571 1117057.1 985439.6977 1208296.89 1219605.638 Glutaric acid 0 10317057.22 13498421.89 12828858.6 11038602.15 9517954.763 14506850.06 11050103.46 Adipic acid 38875952.76 39328502.34 37201199.06 56801083.91 50827057.47 41141345.95 73907915.33 79351246.23 2-Methylglutaric acid 32429547.63 41394012.68 46644298.99 63994404.97 59264035.43 48705952.75 96014774 89289695.53 Uric acid 3858521.63 5583133.906 4174378.367 4046791.204 4716372.2 3184891.326 3479561.069 5551592.335 Glutamic acid 96403826.3 130784103.8 117905458.9 186857758.2 171486903.8 119422392.1 310519541.2 264665835.6 Mannitol 1909996.192 3529688.99 3208866.597 1633196.132 2769313.921 1635058.841 3537644.795 3497361.826 Vanylglycol 6665815.895 3682754.881 4735570.432 2544647.136 7527076.756 3799868.643 5024600.852 5468767.831 Methylglutaric acid 38948314.83 44061435.09 42588712.06 61799456.67 55183862.94 41195580.28 73886534.22 79676295.32 Aspartic acid 16714929.18 29789576.22 23189091.76 48476684.85 45102248 25056547.18 51649449.71 47548553.57 Galactitol 1909996.192 2198134.373 1920480.28 2224037.474 2572026.072 1185520.488 2622144.489 3689684.378 Stachyose 51613.41484 84635.72284 64439.97974 102605.4177 79531.01507 32419.70877 42579.29949 152114.5428 UDP-glucose 3955261.214 5239699.214 4564091.258 6260954.411 6188824.109 4438528.509 8909777.323 8178180.39 UDP-galactose 3955261.214 5239699.214 4564091.258 6260954.411 6188824.109 4438528.509 8909777.323 8178180.39 Pyroglutamic acid 261582222.4 334129783.1 263539476.7 250196941.9 267874347 162839755.2 286872394 287889698.6 UMP 11474030.76 15729662.65 12567772.86 15468172.25 14704517.39 11490338.95 17762906.92 17702905.42 Uridine 778630.1555 0 0 348857.1869 0 0 417085.1494 0 CMP 2518736.18 3241462.708 2643973.87 3487649.526 3155288.757 2840997.874 4259183.449 3895445.88 Citramalic acid 20806281.35 20326138.32 16288187.8 13972367.37 11311699.73 3139008.278 20186282.76 10183826.99 AICAR-P 2768618.453 3078027.603 2963639.4 4784426.151 4123846.25 3161247.173 5818846.444 5292506.852 Galactose 14881164.86 21330961.43 10998563.22 28751867.86 30915625.37 19477814.5 36641666.56 35479558.92 Itaconic acid 959962.5491 1758083.924 1166247.087 2591593.032 2126241.779 1599502.831 3710422.723 2665177.585 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity Pyruvic acid 4.2 87.0088 1060 C00022 Negative 3-Methyl-2-oxovaleric acid 2.5 129.0557 47 C00671 Negative GTP 12 521.9834 135398633 C00044 Negative Lactate 5.16 89.0244 107689 C00186 Negative Malic acid 9.5 133.0143 222656 C00149 Negative GDP 10.9 442.0171 135398619 C00035 Negative Fumaric acid 9.5 115.0037 444972 C00122 Negative Ribitol 6.7 151.0612 6912 C00474 Negative ADP-ribose 8.7 558.0644 33576 C00301 Negative Arabitol 6.7 151.0612 94154 C01904 Negative Maltose 8.4 341.1089 439341 C00897 Negative Lactose 8.3 341.1089 6134 C01970 Negative Cellobiose 8.3 341.1089 10712 C06422 Negative UDP-N-acetylglucosamine 9 606.0743 445675 C00043 Negative IMP 9.3 347.0398 135398640 C00130 Negative Glucosamine 6-phosphate 9 258.0384 439217 C00352 Negative ATP 10 505.9885 5957 C00002 Negative UDP 10 402.9949 6031 C00015 Negative UTP 11 482.9613 6133 C00075 Negative Adenosine 3',5'-diphosphate 10.71 426.0221 159296 C00054 Negative CDP 10.2 402.0109 6132 C00112 Negative GDP-mannose 11 604.0699 135398627 C00096 Negative Butyric acid 8.6 87.0452 264 C00246 Negative Monomethyl glutaric acid 8.89 145.0506 73917 C02930 Negative UDP-glucuronic acid 11.8 579.027 17473 C00167 Negative N-Acetylaspartic acid 8.5 174.0408 65065 C01042 Negative Xylitol 6.7 151.0612 6912 C00379 Negative Mannose 6-phosphate 10.17 259.0224 439198 C00275 Negative Homovanillic acid 4.74 181.0506 1738 C05582 Negative CDP-Ethanolamine 9.5 445.0531 123727 C00570 Negative Fructose 6-phosphate 9.5 259.0224 69507 C00085 Negative Glucose 1-phosphate 9.5 259.0224 65533 C00103 Negative Galactose 1-phosphate 10.03 259.0224 123912 C00446 Negative Glutaric acid 9.23 131.035 743 C00489 Negative Adipic acid 8.89 145.0506 196 C06104 Negative 2-Methylglutaric acid 8.67 145.0506 12046 0 Negative Uric acid 7.1 167.0211 1175 C00366 Negative Glutamic acid 8.5 146.0459 33032 C00025 Negative Mannitol 7.5 181.0718 6251 C00392 Negative Vanylglycol 3.4 183.0663 10805 C05594 Negative Methylglutaric acid 8.74 145.0506 12284 0 Negative Aspartic acid 8.6 132.0302 5960 C00049 Negative Galactitol 7.5 181.0718 11850 C01697 Negative Stachyose 10.05 665.2146 439531 C01613 Negative UDP-glucose 9.6 565.0477 8629 C00029 Negative UDP-galactose 9.9 565.0477 18068 C00052 Negative Pyroglutamic acid 5.6 128.0353 7405 C01879 Negative UMP 9 323.0286 6030 C00105 Negative Uridine 4.8 243.0623 6029 C00299 Negative CMP 9.4 322.0446 6131 C00055 Negative Citramalic acid 9 147.0299 441696 C00815 Negative AICAR-P 8.5 337.0555 65110 C04677 Negative Galactose 9.69 179.0561 439357 C00984 Negative Itaconic acid 9 129.0193 811 C00490 Negative METABOLITES_END #END