#METABOLOMICS WORKBENCH spinelli_lab_20251112_141419 DATATRACK_ID:6676 STUDY_ID:ST004396 ANALYSIS_ID:AN007348 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 20, 2025, 5:29 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 Intracellular polar metabolites profiling on FXS versus TD Lymphoblastoid cell ST:STUDY_TITLE lines (LCL). ST:STUDY_SUMMARY Extract and measure polar metabolites from Lymphoblastoid cell lines (LCL) from ST:STUDY_SUMMARY a individual with Fragile X Syndrome and from one typically developing male ST:STUDY_SUMMARY control. 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 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20250819_TD_Sneha_LCL_FXSvsControl_MK_01.raw SUBJECT_SAMPLE_FACTORS - FXS3 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20250819_TD_Sneha_LCL_FXSvsControl_MK_02.raw SUBJECT_SAMPLE_FACTORS - TD4 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20250819_TD_Sneha_LCL_FXSvsControl_MK_05.raw SUBJECT_SAMPLE_FACTORS - TD6 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20250819_TD_Sneha_LCL_FXSvsControl_MK_06.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 For extraction on cell pellets from in-vitro cultures, samples were incubated on SP:SAMPLEPREP_SUMMARY dry ice with cold 80% methanol. The extracts were spun down and the supernatant SP:SAMPLEPREP_SUMMARY was vacuum dried 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 FXS3 TD4 TD6 Factors Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD 2',3' cyclic AMP 288768.223 108336.375 198632.659 143649.868 2-Hydroxybutyric acid 658867.759 616526.435 493585.214 556028.745 3',5' cyclic AMP 288768.223 108336.375 198632.659 143649.868 3-Hydroxymethylglutaric acid 6795145.74 8602341.67 7016924.46 6922224.6 3-Methyl-2-oxovaleric acid 1338897.4 3711153.86 1887644.45 4148114.49 3-Phosphoglyceric acid 10746095.6 21398415.3 16439007.7 13105291.4 4,5-Dihydroorotic acid 272163.34 516949.085 726168.563 700343.942 4-Hydroxybenzaldehyde 5877567.45 5221948.65 8275958.82 4236761.88 4-Pyridoxic acid 583520.489 899497.493 1411399.21 1208495.8 5-Methylcytidine 287853.068 227708.928 254284.625 282838.083 ADP 23529405.4 18389412.2 26718684.8 18171308.8 ADPglucose 1599511.01 895127.593 1404505.85 986066.132 ADPribose 542504.687 1429617 1031329.31 748623.733 Allantoin 9846894.06 7077099.99 9996273.59 11811144.6 AMP 11380860.1 9579628.75 25509561.6 13687017.4 Arabitol 3710883.98 2908360.78 3756084.96 3011550.79 Aspartic acid 145485218 187702229 345966904 300731699 ATP 242397960 217263447 238317240 229073795 Azelaic acid 765087.363 904710.33 1639448.16 1223869.49 CDP 1417758.9 1539408.12 2498825.19 1466357.52 CDP Ethanolamine 1153580.11 1138523.66 6165878.45 1340428.96 Cellobiose 3453605.87 3421374.6 7387507.64 5966526.46 Citramalic acid 583960.88 780396.324 1771344.48 1121579.91 CMP 116428.24 84838.9418 207442.72 166439.32 CTP 10758411.2 16720763.2 17010701.3 16047398.2 dATP 365685.486 339533.484 151149.287 263540.898 Deoxycytidine 980501.969 462925.53 616258.146 564614.856 Dihydroxyacetone phosphate 16610924 12407080.9 13323931.3 12240147 DTagatose 37211765.8 74391485.5 67841164.4 65445002.8 dTDPglucose 71243.395 50206.9478 52975.5539 66350.1792 dTMP 594552.749 1167473.57 937831.852 1109014.31 dUMP 14537.4672 15092.7927 62895.2648 21997.5231 Fructose 1,6-bisphosphate 11393170 22266253.4 15663777.5 17692655.8 Fructose 6-phosphate 4009555.45 15374773.4 7681777.92 4994453.17 Fumaric acid 17570279.3 47064406.5 19168565.9 31971440.5 Galactitol 13557661.3 14245706.5 21532268.3 19290667.9 Galactose 37211765.8 74391485.5 67841164.4 65445002.8 Galactose 1-phosphate 3066753.53 1821921.6 4927782.81 2750065.49 gamma-Aminobutyric acid 35145447.3 50197244.5 68927214.4 57538555.2 GDP 2546150.69 2406455.1 2714369.42 2124971.04 GDP-mannose 1849957.44 1025918.14 908885.51 896684.644 Gluconic acid 10067140.6 2866320.4 8597893.12 8008280.67 Glucosamine 6-phosphate 467804.187 275971.166 525698.761 358297.752 Glucose 37211765.8 74391485.5 67841164.4 65445002.8 Glucose 1-phosphate 3066753.53 1821921.6 4927782.81 2752226.9 Glucose 6-phosphate 3066753.53 1821921.6 4927782.81 2718202.3 Glucuronic acid 184419.656 157489.135 223503.651 254092.913 Glutamic acid 497985363 724650919 964906592 831658130 Glyceric acid 847709.483 1075654.5 1224990.95 1473324.85 Glycerol 2344799.49 2435666.81 4524846.16 2746741.28 GMP 679286.797 667305.002 2360017.19 1178239.98 Guanosine 90090.5579 262988.909 265155.671 328012.816 Homovanillic acid 6179060.57 7115984.27 19634247.5 7964653.75 Hydroxyphenyllactic acid 6179060.57 7115984.27 19634247.5 7964653.75 IMP 995879.788 1772601.57 4347574.44 1365718.2 Lactic acid 1503235129 552513084 392696790 513556541 Lactose 3543743.5 3421374.6 7387507.64 5966526.46 Maleic acid 2614073.91 2848063.1 5144056.09 4746237.04 Malic acid 224966099 591872029 234946747 400826037 Malonic acid 1728387.01 1866554.73 2501216.94 3067920 Maltose 3291570.91 3421374.6 7387507.64 5966526.46 Mannitol 13557661.3 14245706.5 21532268.3 19290667.9 Mannose 6-phosphate 3066753.53 1821921.6 4927782.81 2965129.77 Melibiose 3320194.09 3421374.6 7387507.64 5966526.46 Mesotartaric acid 15446375.6 6803726.89 17036134.7 1700429.41 Methylmalonic acid 5192482.94 10046737.4 14136104.5 9139669.5 N-Acetylalanine 3201521.72 5380245.62 4769287.44 5106703.58 N-Acetylaspartic acid 47427820 9665675.3 28726150.9 15266889.2 N-Acetylcysteine 447186.919 563154.37 219274.075 739118.429 N-Acetylglutamic acid 9461132.55 11828525.2 14524271.1 13988633.8 N-Acetylmethionine 10185294.4 22881205.8 14684412.1 21666247.6 N-Acetylserine 50808743.1 83905341.7 83478717.7 83615933 NADH 3023445.01 3043470.83 2789578.59 2337207.86 N-Formylmethionine 9153800.23 8069301.16 10739873.5 15342152.6 N-Methyl-D-aspartic acid 497985363 724650919 964906592 831658130 O-Acetylserine 50808743.1 83905341.7 83478717.7 83615933 Orotic acid 317213.876 1309827.2 870724.988 1961473.26 Oxalic acid 5478801.9 7887281.16 12041205.8 7420728.54 Oxaloacetic acid 342268.395 672122.969 950632.145 545265.341 Palatinose 3347089.84 3421374.6 7387507.64 5966526.46 Phosphocreatine 1799394.69 561103.581 2202548.51 1548009.19 Phosphoenolpyruvic acid 5008230.72 5088003.16 4089566.56 3068354.71 Psicose 37211765.8 74391485.5 67841164.4 65445002.8 Pyroglutamic acid 750991322 498898918 530583045 626070992 Pyruvic acid 1844275.61 1407488.9 2339596.69 2858628.44 sn-Glycero2phosphate 3275473.64 1149085.13 10962559 2055736.92 sn-Glycero3phosphate 3275473.64 1149085.13 10962559 2055736.92 Sorbitol 13557661.3 14245706.5 21532268.3 19290667.9 Sorbose 37211765.8 74391485.5 67841164.4 65445002.8 Succinic acid 3699982.15 7288564.68 9040744.9 7871930.86 Sucrose 3321045.83 3421374.6 7387507.64 5966526.46 Tartaric acid 16745031.8 7843756.21 18853644.9 1997673.8 Trehalose 3376896.93 3421374.6 7387507.64 5966526.46 UDP 291440.225 454524.735 879163.837 353439.534 UDP-galactose 68024179.6 83575928.1 89903138.6 79432801.6 UDP-N-acetylglucosamine 101359691 90951127.9 84261523.1 75989142.9 Ureidopropionic acid 25039591 39797663.6 38708978.8 49688650 Uric acid 22523678.3 9767960.93 22463768.7 10826431.8 Uridine 312426.781 708828.838 1017307.04 958921.666 UTP 28014568.9 47556358.6 47445842.4 46704996.1 Valeric acid 4085825.83 13801222.8 3456557.68 10708760.5 Xanthosine 368681.288 335355.834 286836.165 294968.432 Xylitol 3710883.98 2908360.78 3756084.96 3011550.79 Xylose 1007201.98 2248113.94 1388147.58 2235852.52 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity 2',3' cyclic AMP 5 328.04524 101812 C02353   Negative 2-Hydroxybutyric acid 5.2 103.04007 11266 C05984 Negative 3',5' cyclic AMP 5 328.04524 6076 C00575 Negative 3-Hydroxymethylglutaric acid 8.3 161.04555 1662 C03761 Negative 3-Methyl-2-oxovaleric acid 2.5 129.05572 47 C00671 Negative 3-Phosphoglyceric acid 10 184.98566 724 C00597 Negative 4,5-Dihydroorotic acid 6.2 157.02548 648 C00337 Negative 4-Hydroxybenzaldehyde 3.4 121.0295 126 C00633 Negative 4-Pyridoxic acid 2.6 182.04588 6723 C00847 Negative 5-Methylcytidine 5.9 256.09389 92918 0 Negative ADP 8.7 426.02214 6022 C00008 Negative ADPglucose 10.3 588.07496 16500 C00498 Negative ADPribose 8.7 558.0644 33576 C00301 Negative Allantoin 7.5 157.03671 204 C01551 Negative AMP 8 346.05581 6083 C00020 Negative Arabitol 6.7 151.0612 94154 C01904 Negative Aspartic acid 8.6 132.03023 5960 C00049 Negative ATP 9.5 505.98847 5957 C00002 Negative Azelaic acid 6.2 187.09758 2266 C08261 Negative CDP 10.2 402.0109 6132 C00112 Negative CDP Ethanolamine 9.5 445.0531 123727 C00570 Negative Cellobiose 8.3 341.10893 10712 C06422 Negative Citramalic acid 8.7 147.0299 441696 C00815 Negative CMP 9.4 322.04457 6131 C00055 Negative CTP 10.75 481.97723 6176 C00063 Negative dATP 9.3 489.99355 15993 C00131 Negative Deoxycytidine 5.1 226.08333 13711 C00881 Negative Dihydroxyacetone phosphate 9.5 168.99075 668 C00111 Negative DTagatose 9.4 179.05611 439312 C00795 Negative dTDPglucose 8.86 563.06848 443210 C00842 Negative dTMP 7.5 321.04933 9700 C00364 Negative dUMP 7.8 307.03368 65063 C00365 Negative Fructose 1,6-bisphosphate 11 338.98877 172313 C00354 Negative Fructose 6-phosphate 9.5 259.02244 69507 C00085 Negative Fumaric acid 9.5 115.00368 444972 C00122 Negative Galactitol 7.5 181.07176 11850 C01697 Negative Galactose 9.69 179.05611 439357 C00984 Negative Galactose 1-phosphate 10.03 259.02244 123912 C00446 Negative gamma-Aminobutyric acid 8.5 102.05605 223130 C00334 Negative GDP 10.5 442.01705 135398619 C00035 Negative GDP-mannose 11 604.06988 135398627 C00096 Negative Gluconic acid 7.6 195.05103 10690 C00257 Negative Glucosamine 6-phosphate 9 258.03843 439217 C00352 Negative Glucose 9.5 179.05611 5793 C00267 Negative Glucose 1-phosphate 9.94 259.02244 65533 C00103 Negative Glucose 6-phosphate 10 259.02244 5958 C00092 Negative Glucuronic acid 9.5 193.03538 94715 C00191 Negative Glutamic acid 8.5 146.04588 33032 C00025 Negative Glyceric acid 6.7 105.01933 439194 C00258 Negative Glycerol 5.23 91.04007 753 C00116 Negative GMP 9.9 362.05072 135398631 C00144 Negative Guanosine 6.5 282.08439 135398635 C00387 Negative Homovanillic acid 4.74 181.05063 1738 C05582 Negative Hydroxyphenyllactic acid 4.9 181.05063 9378 C03672 Negative IMP 9.5 347.03982 135398640 C00130 Negative Lactic acid 5.36 89.02442 107689 C00186 Negative Lactose 8.3 341.10893 6134 C01970 Negative Maleic acid 8.67 115.00368 444266 C01384 Negative Malic acid 9.5 133.01425 222656 C00149 Negative Malonic acid 9.5 103.00368 867 C00383 Negative Maltose 8.4 341.10893 439341 C00897 Negative Mannitol 7.5 181.07176 6251 C00392 Negative Mannose 6-phosphate 10.17 259.02244 439198 C00275 Negative Melibiose 8.4 341.10893 440658 C05402 Negative Mesotartaric acid 10.34 657.73469 447315 C00552 Negative Methylmalonic acid 8.8 117.01933 487 C02170 Negative N-Acetylalanine 4.85 130.05097 88064 0 Negative N-Acetylaspartic acid 8.5 174.0408 65065 C01042 Negative N-Acetylcysteine 4.2 162.02304 12035 C06809 Negative N-Acetylglutamic acid 8.2 188.05645 70914 C00624 Negative N-Acetylmethionine 3.2 190.05434 6180 C02712 Negative N-Acetylserine 6 146.04588 65249 0 Negative NADH 7.7 664.11749 439153 C00004  Negative N-Formylmethionine 3.55 176.03869 439750 C03145 Negative N-Methyl-D-aspartic acid 8.5 146.04588 22880 C12269 Negative O-Acetylserine 5.94 146.04588 99478 C00979 Negative Orotic acid 5.8 155.00983 967 C00295 Negative Oxalic acid 10.4 88.98803 971 C00209 Negative Oxaloacetic acid 9 130.9986 970 C00036 Negative Palatinose 8.5 341.10893 439559 C017424 Negative Phosphocreatine 9 210.02853 587 C02305 Negative Phosphoenolpyruvic acid 10.2 166.9751 1005 C00074 Negative Psicose 9.6 179.05611 90008 C06468 Negative Pyroglutamic acid 5.6 128.03532 7405 C01879 Negative Pyruvic acid 4.5 87.00877 1060 C00022 Negative sn-Glycero2phosphate 8.65 171.0064 2526 C02979 Negative sn-Glycero3phosphate 8.7 171.0064 439162 C00093 Negative Sorbitol 7.5 181.07176 5780 C00794 Negative Sorbose 9.5 179.05611 6904 C01452 Negative Succinic acid 8.8 117.01933 1110 C00042 Negative Sucrose 8.31 341.10893 5988 C00089 Negative Tartaric acid 10.1 149.00916 444305 C00898 Negative Trehalose 8.3 341.10893 7427 C01083 Negative UDP 9.5 402.99492 6031 C00015 Negative UDP-galactose 9.9 565.04774 18068 C00052 Negative UDP-N-acetylglucosamine 9 606.07429 445675 C00043 Negative Ureidopropionic acid 8.1 131.04622 111 C02642 Negative Uric acid 7.1 167.02106 1175 C00366 Negative Uridine 4.8 243.06226 6029 C00299 Negative UTP 10.6 482.96125 6133 C00075 Negative Valeric acid 3.01 101.0608 7991 C00803 Negative Xanthosine 6.7 283.06841 64959 C01762 Negative Xylitol 6.7 151.0612 6912 C00379 Negative Xylose 6.97 149.04555 135191 C00181 Negative METABOLITES_END #END