#METABOLOMICS WORKBENCH spinelli_lab_20251112_110517 DATATRACK_ID:6667 STUDY_ID:ST004390 ANALYSIS_ID:AN007336 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 20, 2025, 4:30 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 Richter 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 Human Fragile X Syndrome cortex compared to typically developing ST:STUDY_SUMMARY Polar metabolic profiling of human cortex with and without Fragile X Syndrome. ST:INSTITUTE UMass Chan Medical School ST:LAST_NAME UMass Chan ST:FIRST_NAME Richter 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 Human 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 - TD01 Sample source:Homo Sapien Male Cortex | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_17.raw SUBJECT_SAMPLE_FACTORS - TD02 Sample source:Homo Sapien Male Cortex | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_18.raw SUBJECT_SAMPLE_FACTORS - TD03 Sample source:Homo Sapien Male Cortex | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_19.raw SUBJECT_SAMPLE_FACTORS - TD04 Sample source:Homo Sapien Male Cortex | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_20.raw SUBJECT_SAMPLE_FACTORS - TD05 Sample source:Homo Sapien Male Cortex | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_21.raw SUBJECT_SAMPLE_FACTORS - TD06 Sample source:Homo Sapien Male Cortex | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_22.raw SUBJECT_SAMPLE_FACTORS - FXS01 Sample source:Homo Sapien Male Cortex | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_23.raw SUBJECT_SAMPLE_FACTORS - FXS02 Sample source:Homo Sapien Male Cortex | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_24.raw SUBJECT_SAMPLE_FACTORS - FXS03 Sample source:Homo Sapien Male Cortex | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_25.raw SUBJECT_SAMPLE_FACTORS - FXS04 Sample source:Homo Sapien Male Cortex | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_26.raw SUBJECT_SAMPLE_FACTORS - FXS05 Sample source:Homo Sapien Male Cortex | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_27.raw SUBJECT_SAMPLE_FACTORS - FXS06 Sample source:Homo Sapien Male Cortex | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20230214_RichterLab_SS_28.raw #COLLECTION CO:COLLECTION_SUMMARY The post-mortem brain tissues were powdered under liquid nitrogen and samples CO:COLLECTION_SUMMARY were normalized based on tissue weight. The metabolites were extracted using 80% CO:COLLECTION_SUMMARY methanol. CO:SAMPLE_TYPE Brain cortex #TREATMENT TR:TREATMENT_SUMMARY Frozen post-mortem cortex and cerebellum tissues were obtained from University TR:TREATMENT_SUMMARY of California, Davis Brain Repository for Typically Developing (TD) males and TR:TREATMENT_SUMMARY age-matched FXS males (n=6 each genotype). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The metabolites were extracted using 80% methanol. The extracts were spun down SP:SAMPLEPREP_SUMMARY and the supernatant 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 TD01 TD02 TD03 TD04 TD05 TD06 FXS01 FXS02 FXS03 FXS04 FXS05 FXS06 Factors Sample source:Homo Sapien Male Cortex | Genotype:TD Sample source:Homo Sapien Male Cortex | Genotype:TD Sample source:Homo Sapien Male Cortex | Genotype:TD Sample source:Homo Sapien Male Cortex | Genotype:TD Sample source:Homo Sapien Male Cortex | Genotype:TD Sample source:Homo Sapien Male Cortex | Genotype:TD Sample source:Homo Sapien Male Cortex | Genotype:FXS Sample source:Homo Sapien Male Cortex | Genotype:FXS Sample source:Homo Sapien Male Cortex | Genotype:FXS Sample source:Homo Sapien Male Cortex | Genotype:FXS Sample source:Homo Sapien Male Cortex | Genotype:FXS Sample source:Homo Sapien Male Cortex | Genotype:FXS 2-Phosphoglyceric acid 9250075.704 34091689.75 14147359.92 16368313.19 4332033.987 15277173.51 15860308.21 28548141.58 13437518.5 24260532.39 25169957.17 27483816.39 3-Phosphoglyceric acid 9250075.704 34091689.75 14147359.92 16368313.19 4332033.987 15277173.51 15860308.21 28548141.58 13437518.5 24260532.39 25169957.17 27483816.39 Adenosine 149941.9845 46698.9152 2511.694061 17993.41662 45858.545 8501.094039 78850.75452 271103.5207 309172.6469 33384.83488 155048.5912 82880.1325 ADP 40507716.72 23177219.01 15587256.23 22814397.71 25826694.96 15905103.21 37102782.75 14125273.82 28311049.08 11818875.84 37583511.43 21177134.73 ATP 9638598.597 5674041.975 3895131.418 7480785.843 4316355.344 3753625.922 14197683.18 5375084.812 9402896.076 5593402.186 9184374.358 11111405.66 AMP 114686115 27639352.48 43798686.93 31678525.72 50290999.77 43224983.87 78100484.22 31471843.83 59235872.47 15477036.87 137398451 18511354.32 Azelaic acid 1765663.28 7502612.466 3548561.951 3740507.639 4871576.05 2517602.599 1737179.836 5815620.939 2264048.742 2701049.347 2639770.074 2524539.857 NADH 9817267.194 15851042.98 7877402.851 7503574.259 10111688.67 13476598.81 12750975.42 9121914.729 20496274.26 6262689.684 20516735.82 9344787.508 Citric acid 145278373.7 141978953.8 55297527.61 191056156.4 123798984.8 67194664.29 57049109.58 71342901.96 141865526.1 204512515.5 229587932.6 68455890.63 3',5' cyclic AMP 1087120.229 1497574.785 399483.4528 1205706.143 1631287.453 1057627.835 1193116.049 819090.4109 961806.1863 420695.808 497173.3408 898029.7063 CDP 470215.6139 33136.44111 24411.94248 73860.24722 110506.6307 26403.09027 299843.8368 38925.29391 179587.3875 136699.6805 56078.46088 77231.2718 CMP 7239627.698 4885879.401 1607820.025 1789220.508 2768977.219 1932625.994 4370362.013 5462102.222 3362397.173 1617888.608 3067718.334 1601240.536 CTP 36858.6777 2414.914176 0 15229.6501 7277.137545 0 27029.45894 1873.095939 14658.23843 27871.45367 804.440316 46617.5735 beta-D-sedoheptulose 3449254.304 15463309.08 14391460.9 14330817.43 11010876.4 9864458.462 4770977.935 4064054.343 8355813.718 7208971.148 5052620.884 4459397.845 Fumaric acid 26080275.84 43000673.29 24325442.92 59213538.88 26610919.39 21214792.75 25000571.99 28899628.52 32259883.69 22987984.86 30470564.93 24267096.94 Galactose 372020173 335587780.2 108857602.4 196780701.2 237468885.4 171652432.5 236203272.6 159633745.5 187774662.5 131374254.5 191035561.2 172358186.8 Gluconic acid 57359273.5 295354744.9 762510723.6 376480071.5 370239185.6 185535445.4 59265919.18 187461959.9 226238059.8 104917082.7 81258154.65 59827043.61 Glucosamine 6-phosphate 39917276.09 154248081.3 43834166.68 54989229.18 35048353.8 82575219.34 49330758.85 34906388.52 43221899.33 36870821.05 78488363.29 15277328.03 Glutamic acid 722930334.5 836554152.6 379114641.3 779366331.3 831851697.6 585754142.6 641612516.7 678071690.6 698010673.8 466765302.8 914149738.7 683332850.8 Glycerol 14358452.9 10946957.58 6004973.023 9108069.213 10971706.88 6362963.973 9707462.315 6364994.606 9531073.973 4022302.976 4995748.473 10342857.31 sn-Glycero-3-phosphate 111570719 13619838.87 10546473.93 14461176.95 41607201.38 8482592.631 22806483.66 42807829.88 16703312.31 4357581.222 11970394.93 23113941.11 Guanosine 53444199.49 23677613.94 9010985.612 7875024.399 20583551.4 29342445.88 54375211.94 32170496.87 44423541.38 80653773.98 68305900.3 25489977.11 Isocitric acid 145166765.2 141712474.2 54937153.85 188273879.3 123260139.4 66878890.53 56697001.32 71606773.93 141514019.9 204138184.4 228099507.1 72269202.21 Itaconic acid 7314760.759 9183387.123 7392314.432 11841402.39 11068139.19 8224715.324 7956306.968 6712816.553 4943199.637 7327327.47 9222878.757 6273937.506 Lactate 2557759217 4456019631 2474946989 3197906926 3400707555 2797535663 2594565981 3345786117 2872991869 1874479675 2890349239 1601606260 Malic acid 206995191.8 362679009.3 208477336.8 490698295.3 215211719.4 180568505.5 206707136.4 241666868.7 264925687.2 192951097.7 254593134.9 200649689.8 Mannitol 73233235.31 138092110.7 53620095.76 60085273.58 78803480.61 75094779.39 37536131.76 87132936.2 84442369.79 57274124.33 96372735.34 29052863.53 Methylmalonic acid 82794709.79 77569259.72 25043944.72 58045101.16 78067676.59 50421861.98 53158310.72 66556509 54185078.58 39370947.82 29859451.1 48780674.62 N-Acetylaspartic acid 5001223847 4602260895 2743546990 3093985345 4197297569 3666639610 5221485243 4213093156 4811719677 4092497816 5447032032 4180327724 Orotic acid 622618.7531 611726.8736 192337.0465 905212.9129 370500.9168 353600.9471 687438.2916 1149618.334 758578.48 599470.742 893628.2628 966076.2286 Phosphocreatine 0 7745.246643 7230.332372 0 0 0 17687.75994 0 0 0 0 0 Phosphoenolpyruvic acid 3703239.419 5702711.716 1539474.777 3065987.318 1682236.116 2524595.827 5037878.501 5432992.851 5156436.278 4255531.097 5029393.724 6135980.229 Pyruvic acid 530058.078 1840111.913 834882.0147 1115506.828 747204.1208 861302.444 1445578.226 1276008.964 1277272.702 1640299.228 2745929.239 607865.1875 Succinic acid 91316403.16 85363683.31 28588682.74 63692469.4 85552138.73 56511273.78 58748251.57 72411571.98 59919397.11 43136571.11 32910379.59 54149242.39 Uracil 81107378.91 26821652.29 29006611.08 51245978.85 64993783.51 25874738.53 51577196.67 32036926.47 47790551.86 16359517.48 18269146.6 56236128.23 Uric acid 45069373.02 149728130.8 193228796.4 179518742.8 155979876.6 85795000.38 33976156.99 144335208.7 42436158.61 80905906.4 69854853.61 46298978.15 Uridine 37857407.49 81599909.07 37196600.86 41507609.34 25682121.52 45084307.25 34259431.18 31965236.53 30235554.85 28490363.32 30481723.99 38711374.28 UDP 4419053.714 2128653.56 976295.3637 1184239.003 1090309.378 1378446.846 4109202.339 2066427.987 2844152.446 1615970.734 5227994.299 1127770.967 UMP 14659659.74 3278638.017 1752135.325 1703360.955 4262945.33 2918723.056 6206172.165 3283524.614 4690313.578 1006589.563 9818019.48 811262.9029 UTP 389244.8928 54805.55858 30691.4849 96397.57736 55899.84153 32816.87055 453763.8593 93653.98079 193775.2324 151689.593 116188.1782 276326.8581 Xanthosine 54878778.14 13298485.97 7962824.776 12566197.08 22591841.93 12872408.36 19089185.1 8964042.522 13653789.76 5784100.088 8274316.269 18333305.21 Xylose 7815310.016 7998170.744 3127236.258 5673223.762 4977799.742 3627605.81 5953142.011 25946575.91 8233574.759 5150932.548 4991459.546 4245759.424 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID 2-Phosphoglyceric acid 10.2 184.9857 59 C00631 3-Phosphoglyceric acid 10 184.9857 724 C00597 Adenosine 4.4 266.0895 60961 C00212 ADP 8.9 426.0221 6022 C00008 ATP 10 505.9885 5957 C00002 AMP 8 346.0558 6083 C00020 Azelaic acid 6.2 187.0976 2266 C08261 NADH 7.7 664.1175 439153 C00004 Citric acid 10.9 191.0197 311 C00158 3',5' cyclic AMP 5 328.0452 6076 C00575 CDP 10.2 402.0109 6132 C00112 CMP 9.4 322.0446 6131 C00055 CTP 11.3 481.9772 6176 C00063 beta-D-sedoheptulose 7.5 209.0667 441483 C08355 Fumaric acid 9.5 115.0037 444972 C00122 Galactose 9.69 179.0561 439357 C00984 Gluconic acid 7.8 195.051 10690 C00257 Glucosamine 6-phosphate 9 258.0384 439217 C00352 Glutamic acid 8.5 146.0459 33032 C00025 Glycerol 5.23 91.0401 753 C00116 sn-Glycero-3-phosphate 8.65 171.0064 439162 C00093 Guanosine 6.5 282.0844 135398635 C00387 Isocitric acid 10.8 191.0197 1198 C00311 Itaconic acid 9 129.0193 811 C00490 Lactate 5.16 89.0244 107689 C00186 Malic acid 9.5 133.0143 222656 C00149 Mannitol 7.5 181.0718 6251 C00392 Methylmalonic acid 8.8 117.0193 487 C02170 N-Acetylaspartic acid 8.5 174.0408 65065 C01042 Orotic acid 5.8 155.0098 967 C00295 Phosphocreatine 9 210.0285 587 C02305 Phosphoenolpyruvic acid 10.6 166.9751 1005 C00074 Pyruvic acid 4.2 87.0088 1060 C00022 Succinic acid 8.8 117.0193 1110 C00042 Uracil 4.7 111.02 1174 C00106 Uric acid 7.1 167.0211 1175 C00366 Uridine 4.8 243.0623 6029 C00299 UDP 10 402.9949 6031 C00015 UMP 9 323.0286 6030 C00105 UTP 11 482.9613 6133 C00075 Xanthosine 6.7 283.0684 64959 C01762 Xylose 6.97 149.0456 135191 C00181 METABOLITES_END #END