#METABOLOMICS WORKBENCH spinelli_lab_20251112_132543 DATATRACK_ID:6674 STUDY_ID:ST004395 ANALYSIS_ID:AN007346 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 20, 2025, 5:25 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 Extracellular polar metabolites profiling from Lymphoblastoid cell lines (LCL) ST:STUDY_TITLE from individuals with Fragile X Syndrome versus from typically developing male ST:STUDY_TITLE controls. ST:STUDY_SUMMARY Extract and measure polar metabolites from media used to grow Lymphoblastoid ST:STUDY_SUMMARY cell lines (LCL) from individuals with Fragile X Syndrome versus from typically ST:STUDY_SUMMARY developing male 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:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_FXS101.raw SUBJECT_SAMPLE_FACTORS - FXS1_02 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_FXS102.raw SUBJECT_SAMPLE_FACTORS - FXS1_03 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_FXS103.raw SUBJECT_SAMPLE_FACTORS - FXS2_01 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_FXS201.raw SUBJECT_SAMPLE_FACTORS - FXS2_02 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_FXS202.raw SUBJECT_SAMPLE_FACTORS - FXS2_03 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_FXS203.raw SUBJECT_SAMPLE_FACTORS - TD_01 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_WT01.raw SUBJECT_SAMPLE_FACTORS - TD_02 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_WT02.raw SUBJECT_SAMPLE_FACTORS - TD_03 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Media_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)). Media was collected from the well CO:COLLECTION_SUMMARY with growing cells upon extraction of the cell pellet. CO:SAMPLE_TYPE Media #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 media polar metabolites extraction, 10ul of sample was added to 90ul of 80% SP:SAMPLEPREP_SUMMARY methanol and vortexed for 10mins before spinning to collect the supernatant SP:SAMPLEPREP_SUMMARY containing extracted metabolites and vacuum dried. The extract was then SP:SAMPLEPREP_SUMMARY resuspended 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 TD_03 Factors Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD Sample source:Extracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD 3-Hydroxymethylglutaric acid 138674996 89755436.37 94438381.99 162855053.5 160698528.9 138192470.2 352705081.8 277675655.3 310903762.7 3-Methyl-2-oxovaleric acid 79505560.1 67682596.46 67347313.53 111266880 95167660.14 82393033.28 62835912.66 53725749.84 62187329.22 3E-Methylglutaconic acid 28105008 17693520.34 16289511.08 31535051.32 29173437.54 25518426.03 73177880.44 68917673.89 78621387.2 Vanylglycol 7888591.94 4841776.277 9493106.445 7563960.357 6217781.371 7022784.234 6469757.199 5432363.464 6037817.825 Imidazoleacetic acid 23853090.9 23807867.21 20493393.36 25884516.92 20658285.05 19155718.74 30801986.09 22964073.85 32633754.54 ADP-ribose 65532.9072 279547.671 255277.0726 127080.9871 0 28633.10137 0 0 0 AMP 2861967.9 7090563.426 6560275.104 2911493.378 2958872.379 1666911.503 1398151.291 960815.555 862354.9747 AICAR-P 9488790.82 9690101.792 9592413.465 10105358.14 10331912.35 9773202.316 7290813.324 8210610.736 7908843.596 Allantoin 67009244.3 73662619.98 68925353.7 83963893.85 70441134.67 63983592.07 80755898.26 76934611.68 82200249.92 Allose 51227714.3 25351634.11 26752590.46 60634432.79 57832745.19 51409122.65 152890068.4 146069440.5 161084801.3 Azelaic acid 48674675.9 24005017.25 25179322.16 57572392.84 55297924.61 49239478.24 145927430.3 141496803.7 154938983.2 Arabitol 125992261 125367358.6 123711945 149985302.8 131299834.6 119220261.5 150056671.7 138304727.7 148399991.9 Vitamin C 14538435.9 14829592.78 14726996.62 32148820.41 32628345.36 22212865.67 8006304.242 7932847.777 9688192.438 Aspartic acid 172007765 154703150.7 170215898.6 235618184 195752288.5 181008364.1 267772920 259068739.9 271858360.7 Butyric acid 10315360.5 6138568.627 5384878.173 13554602.32 11539062.56 7558404.432 25291665.57 21146716.3 23716010.18 Cellobiose 369745.742 509489.6301 484749.7607 541796.9951 422487.2046 781539.4336 700789.5849 431355.2291 346695.8975 Citramalic acid 44861105.7 43110925 47054354.51 68076165.94 55230924.1 49618866.25 38682667.39 37368398.97 43120885.42 Citric acid 132437413 168829893.5 184881605 206203625.6 159761283.8 144358026.4 141910437.6 130732674 147947202.1 Cysteine 11014598.1 13351510.21 13332937.31 18380043.14 15020368.4 9955632.996 19461743.5 18049398.25 17610104.88 CDP 32511.2474 66457.99352 64527.54504 53161.35741 42688.9574 21928.5281 24250.69171 5726.429566 0 Folic acid 2602924.17 2651442.437 2777207.989 3542190.31 2735740.595 2645817.151 3360746.158 3153264.992 3559383.852 Fumaric acid 19977044.6 26459033.22 24780594.18 30655996.45 25559144.93 20888974.36 18481612.5 17595130.03 19833647.23 Galactitol 148573833 151554024.3 149137430.5 161998344.4 152703899.4 138545534.3 173846922.6 161849366.6 186253277.5 Galactose 263114161 266413317 283416911.5 331468324.1 244329216.3 227946228.7 331301719.8 309652206.9 369484190.5 Gluconic acid 39959301.9 40248161.34 44207122.48 54503504.34 52133022.45 45273754.31 53941824.34 53373544.32 52617967.04 Glucosamine 6-phosphate 485872.71 559415.9801 429083.2627 916306.3004 677434.4795 536524.9693 375577.6992 348383.1485 412778.3954 Glucose 6-phosphate 873145.422 1113249.434 1025002.114 1018423.101 973922.6332 1056916.382 748569.8803 755400.2048 1157475.832 Glutamic acid 795605548 781871918 821089846.4 1169612739 997836265 898098144.8 704111863.5 649092687.3 744258522.4 Glutaric acid 135744841 139299995.7 149717688.9 155944873.6 129885601.6 119246258.7 193510807.3 193647470 209652595.3 GDP 18774.929 41107.28494 35342.42592 13312.473 10625.88397 3522.58083 17272.11757 1777.098761 3445.660934 GDP-mannose 0 13511.45363 13353.15058 1637.904984 0 1891.595179 0 3636.860621 0 Homovanillic acid 35914989.7 40749608.96 42180182.92 71078123.96 57366360.27 54845397.04 36992003.41 37542753.72 41616692.8 Itaconic acid 8574453.49 8902143.782 9215728.972 14316042.59 12123179.73 10058543.75 6627562.449 6461444.126 6877641.831 Lactate 14250000000 17604233785 18044092571 16694971044 13052478645 11922783450 7526383274 6548194605 7825460646 Lactose 368993.984 508225.7202 482509.8237 537878.5493 402083.7381 768978.525 694506.5978 446618.577 346695.8975 Malic acid 215077060 254719538.5 248009437.1 307946746.9 259729215.6 237236505 223958168.3 196871625.4 202281161.6 Maltose 363253.656 503781.246 491632.5708 531996.8389 416491.5725 746917.5584 707893.6793 455003.2797 346695.8975 Mannitol 148573833 151554024.3 149137430.5 161998344.4 152703899.4 138545534.3 173846922.6 161849366.6 186253277.5 Mannose 48674675.9 24005017.25 25179322.16 57572392.84 55297924.61 49239478.24 145927430.3 141496803.7 154938983.2 Methylglutaric acid 1708967410 1606051106 1604596774 1753542840 1426569749 1324436717 3126945349 2900345077 2972943767 Methylmalonic acid 210483758 206286365.7 209138897.4 216463846.8 200896107.9 191664952.8 280093619 259297426 279416823.1 N-Acetylaspartic acid 14351381.3 16974119.25 19986587.9 17593769.45 18105086.28 15407013.46 15881745.58 16117458.88 17899654.31 2-Oxoglutaric acid 63703146.8 73943702.22 98302276.85 121190294.2 117494370.9 90138430.44 41690261.82 37163466.09 41757263.63 Pyroglutamic acid 7630179575 7371616618 7572351901 7485744937 6751235479 5813841793 9421613333 8184807812 8957764152 Psicose 51227714.3 25351634.11 26752590.46 60634432.79 57832745.19 51409122.65 152890068.4 146069440.5 161084801.3 Pyruvic acid 106849335 107880033 113287919.1 194932027.4 176028990.4 151131191.3 83638405.44 62543532.78 73534535.48 Ribitol 110480160 113916428.2 114033223.1 138202702.5 115952785.9 110292174.4 136826330.2 138304727.7 136387578.8 Sorbitol 3352107.05 2622819.421 3153144.951 4521018.556 3330043.07 3120618.079 7116702.778 5974202.003 7233378.259 Stachyose 863772.978 895921.1319 916754.8639 1019638.472 886014.8936 779539.1547 1037558.861 915855.0098 998970.0156 Succinic acid 210483758 206286365.7 209138897.4 216463846.8 200896107.9 191664952.8 280093619 259297426 279416823.1 Uric acid 223741175 239208687.8 244796607.9 291409886.4 234936851.2 203839431.2 292149515.4 269650789.8 282273557.7 Uridine 4405957.33 5020790.809 4935490.149 6274314.35 5089021.47 4227048.73 5580280.368 4841703.917 5928246.665 UDP 214305.325 323322.2297 355536.8755 270290.6482 151505.0145 109461.37 76984.52608 138366.701 235940.805 UDP-glucose 181878.527 295819.3476 272885.4204 296580.5794 242463.1346 150200.6301 119989.7357 67636.03648 61171.28766 UDP-N-acetylglucosamine 250687.479 524342.2132 452986.666 309807.7163 270305.4693 179083.3331 43462.9417 27028.4788 12886.39271 UDP-galactose 181878.527 295819.3476 272885.4204 296580.5794 242463.1346 150200.6301 119989.7357 67636.03648 61171.28766 UMP 2020540.34 2242493.187 2538498.267 2428237.212 2399422.782 2122886.022 2457158.091 2320219.805 2522548.676 Xylitol 125992261 125367358.6 123711945 149985302.8 131299834.6 119220261.5 150056671.7 138304727.7 148399991.9 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity 3-Hydroxymethylglutaric acid 8.3 161.0456 1662 C03761 Negative 3-Methyl-2-oxovaleric acid 2.5 129.0557 47 C00671 Negative 3E-Methylglutaconic acid 8 143.035 1551553 0 Negative Vanylglycol 3.4 183.0663 10805 C05594 Negative Imidazoleacetic acid 6.58 125.0357 96215 C02835 Negative ADP-ribose 8.7 558.0644 33576 C00301 Negative AMP 8 346.0558 6083 C00020 Negative AICAR-P 8.5 337.0555 65110 C04677 Negative Allantoin 7.5 157.0367 204 C01551 Negative Allose 7.9 179.0561 12285879 C01487 Negative Azelaic acid 6.2 187.0976 2266 C08261 Negative Arabitol 6.7 151.0612 94154 C01904 Negative Vitamin C 4.9 175.0248 54670067 C00072 Negative Aspartic acid 8.6 132.0302 5960 C00049 Negative Butyric acid 8.6 87.0452 264 C00246 Negative Cellobiose 8.3 341.1089 10712 C06422 Negative Citramalic acid 9 147.0299 441696 C00815 Negative Citric acid 10.9 191.0197 311 C00158 Negative Cysteine 9 120.0125 5862 C00097 Negative CDP 10.2 402.0109 6132 C00112 Negative Folic acid 10.3 440.1324 135398658 C00504 Negative Fumaric acid 9.5 115.0037 444972 C00122 Negative Galactitol 7.5 181.0718 11850 C01697 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 Glucose 6-phosphate 10 259.0224 5958 C00092 Negative Glutamic acid 8.5 146.0459 33032 C00025 Negative Glutaric acid 9.23 131.035 743 C00489 Negative GDP 10.9 442.0171 135398619 C00035 Negative GDP-mannose 11 604.0699 135398627 C00096 Negative Homovanillic acid 4.74 181.0506 1738 C05582 Negative Itaconic acid 9 129.0193 811 C00490 Negative Lactate 5.16 89.0244 107689 C00186 Negative Lactose 8.3 341.1089 6134 C01970 Negative Malic acid 9.5 133.0143 222656 C00149 Negative Maltose 8.4 341.1089 439341 C00897 Negative Mannitol 7.5 181.0718 6251 C00392 Negative Mannose 8.05 179.0561 18950 C00936 Negative Methylglutaric acid 8.74 145.0506 12284 0 Negative Methylmalonic acid 8.8 117.0193 487 C02170 Negative N-Acetylaspartic acid 8.5 174.0408 65065 C01042 Negative 2-Oxoglutaric acid 9.3 145.0143 51 C00026 Negative Pyroglutamic acid 5.6 128.0353 7405 C01879 Negative Psicose 9.6 179.0561 90008 C06468 Negative Pyruvic acid 4.2 87.0088 1060 C00022 Negative Ribitol 6.7 151.0612 6912 C00474 Negative Sorbitol 7.5 181.0718 5780 C00794 Negative Stachyose 10.05 665.2146 439531 C01613 Negative Succinic acid 8.8 117.0193 1110 C00042 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 UDP-glucose 9.6 565.0477 8629 C00029 Negative UDP-N-acetylglucosamine 9 606.0743 445675 C00043 Negative UDP-galactose 9.9 565.0477 18068 C00052 Negative UMP 9 323.0286 6030 C00105 Negative Xylitol 6.7 151.0612 6912 C00379 Negative METABOLITES_END #END