#METABOLOMICS WORKBENCH spinelli_lab_20251112_141419 DATATRACK_ID:6676 STUDY_ID:ST004396 ANALYSIS_ID:AN007347 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 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 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 1-Aminocyclopropanecarboxylic acid 4140713.18 9557776.54 11082750.8 10083460.4 2-Amino-2-deoxy-D-mannose 889320.781 1081880.76 3325731.93 2929023.19 3-Aminoisobutanoic acid 139573201 39967445.7 190468898 72603236 3-Dehydroxycarnitine 105083117 159364713 143510553 145162624 3-Methylhistidine 6113473.03 5838131.76 11438283.5 9863583.43 4-Acetamidobutanoic acid 5841709.45 3157688.55 4691744.86 5390147.84 4-Guanidinobutanoic acid 30379704.4 15448897.4 16616034 15865900.1 5-Amino-levulinic acid 417776486 749096888 727520663 830268935 5-Aminopentanoic acid 7188863.34 6130649.16 9895219.52 5360062.36 5'-Methylthioadenosine 6393384.85 5541644.15 3709467.52 4148722.51 Acetylcholine 8352845.76 12313957.1 10942528 11462078.2 AcetylCoA 97981.7734 425199.186 518761.622 20340.9197 Adenine 6951381 7271310.85 6016248.76 7919377.43 Alanine 73063977 186477936 92024315.5 147726532 Allothreonine 29454263.3 59084869.1 71559756.9 65194015.4 alpha-Aminoadipic acid 3915939.71 3549571.82 3995973.26 4761173.35 Anserine 1597590.6 1303557.33 1441032.6 2110988.86 Argininosuccinic acid 2632319.59 9895000.25 2811871.43 5473077 Asparagine 81464836.2 128943416 122221304 142717819 beta-Alanine 73063977 186477936 92024315.5 147726532 Betaine 745409857 2175035740 3525043028 1974966572 Biotin 3681705.06 2407939.63 2712259.31 3274348.5 CAR 16:0 33066.9024 236196.169 240275.975 97528.8598 CAR 2:0 304903893 341610797 299084213 361081660 Carnitine 709002492 826069025 449802396 706294200 Carnosine 10681624 6757447.16 11347748 14437885.5 CDP choline 1846096.75 790060.923 2117477.32 786322.85 Choline 33602395.8 18754904.2 21294256.6 40709538.3 Choline phosphate 2914358637 5479695210 6491932755 5610180399 cis-4-Hydroxy-D-proline 426035357 758693363 736529100 841305437 Citrulline 11262846.8 6920473.33 12616709.2 11289829.2 Coenzyme A 709001.182 563754.408 252964.745 355240.343 Creatine 2870521607 1323684246 2525349412 1677117241 Creatinine 420786877 373219169 518480995 581189035 Cystathionine 469092.96 1366365.84 403047.463 2472325.55 Cytosine 15040870.7 15814438.3 14589255.6 21163784.4 DAlanine 75867425.1 189457225 93076478.7 151794479 Dethiobiotin 3112467.98 2940481.73 2084611.44 2321223.11 Diethanolamine 75380696 65534092.9 55015533.2 57637315.9 Dihydrouracil 687552.324 465196.897 793719.945 678286.281 Glucosamine 887481.951 1078821.81 3310660.26 2939875.82 Glutamine 2415590965 2184959394 3462987133 3284479588 Glutathione 204346880 405039825 295754253 330675870 Glycine 3857297.05 6229934.14 7999048.48 6738530.79 Guanidoacetic acid 61395415.3 12533871.9 78034016.5 37468694.3 Homoserine 29595663.4 59239027 71176318.8 66355634.6 Hypotaurine 61655540 29099737.6 56754347.1 31562823 Hypoxanthine 10035972.6 11558534.3 12873461.1 7500495.84 Inosine 862734.841 4313037.62 2725421.41 2972356.18 Isoleucine 309740171 216887961 341585004 341371281 Kynurenine 385043.052 238633.094 327974.37 524460.248 Leucine 170102273 136834035 260045641 229002765 Methionine 56341806.5 55429473.3 87882759.6 80942834.5 N-Acetylasparagine 220637.973 288287.703 492908.68 332910.772 N-Acetyl-D-galactosamine 533706.975 921293.811 1122049.21 1016908.95 N-Acetylneuraminic acid 1215711.73 1194523.46 1882462.83 1420912.73 NAD+ 64614320.2 48150653.2 60316920 44469166.2 NADP+ 2082233.62 1630652.43 3262534.85 2101750.17 Niacinamide 97472095.3 186844306 140045886 161274635 N-Methylalanine 14862166.2 13193878.9 19790596.6 17129815.2 Norleucine 147427521 119751594 228088837 198606382 Norvaline 745409857 2175035740 3525043028 1974966572 Ophthalmic acid 58596.6441 28081.6071 868099.734 237781.346 o-Succinylhomoserine 1346709.24 918288.156 749177.839 926383.014 Oxidized glutathione 13314257.6 19941544.4 23986752.3 28625841.4 Pantothenic acid 434588018 293416736 338652678 370091479 Phenylalanine 61266470.3 65232011.6 113927449 99793051.7 Phosphoethanolamine 3514367.65 7158227.41 89410822.3 9594586.72 Proline 7880896606 5284639736 7963710734 5288512104 Pyridoxal 17916955.8 7438134.56 12419213.8 12459763.5 S-Adenosylhomocysteine 2662278.06 1891771.29 2292537.05 2078575.95 S-Adenosylmethionine 13189086.5 8841939.48 7921207.2 9455147.36 Sarcosine 73063977 186477936 92024315.5 147726532 Serine 2709742.53 8207726.81 12930273.9 9658461.13 Taurine 344017365 202072416 312012626 216390758 Thiamine 3163881.9 839487.51 1209541.8 2308691.83 Threonine 32309960.7 63656331.4 77355123 69241797.6 trans-4-Hydroxyproline 411950864 735951848 720139230 820808218 Trigonelline 1510858.16 1387483.1 3363922.78 2508220.63 Tryptophan 9380476.68 11059322.8 17763380.8 18627288.1 Tyrosine 21639100.3 27889524.1 38647326.5 43421408.2 Valine 745409857 2175035740 3525043028 1974966572 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity 1-Aminocyclopropanecarboxylic acid 7.6 102.05496 535 C01234 Positive 2-Amino-2-deoxy-D-mannose 7.81 180.08665 440049 C03570 Positive 3-Aminoisobutanoic acid 8.5 104.07061 64956 C05145 Positive 3-Dehydroxycarnitine 7.16 146.11756 134 C01181 Positive 3-Methylhistidine 6.6 170.0924 64969 C01152 Positive 4-Acetamidobutanoic acid 4.7 146.08117 18189 C02946 Positive 4-Guanidinobutanoic acid 8.1 146.0924 500 C01035 Positive 5-Amino-levulinic acid 7.66 132.06552 137 C00430 Positive 5-Aminopentanoic acid 8.71 118.08626 138 C00431 Positive 5'-Methylthioadenosine 3.3 298.09684 439176 C00170 Positive Acetylcholine 7 147.12538 187 C01996 Positive AcetylCoA 8.1 810.13305 444493 C00024 Positive Adenine 4.6 136.06177 190 C00147 Positive Alanine 7.9 90.05496 5950 C00041 Positive Allothreonine 7.5 120.06552 99289 C05519 Positive alpha-Aminoadipic acid 8.7 162.07608 469 C00956 Positive Anserine 7.4 241.12952 112072 C01262 Positive Argininosuccinic acid 10.22 291.12991 16950 C03406 Positive Asparagine 8.26 133.06077 6267 C00152 Positive beta-Alanine 7.8 90.05496 239 C00099 Positive Betaine 5.6 118.08626 247 C00719 Positive Biotin 4.5 245.09544 171548 C00120 Positive CAR 16:0 2.35 401.34996 11953816 C02990 Positive CAR 2:0 5.5 204.12303 7045767 C02571 Positive Carnitine 7 162.11247 10917 C00318 Positive Carnosine 8.4 227.11387 439224 C00386 Positive CDP choline 8.8 490.12243 13804 C00307 Positive Choline 9.75 105.11482 305 C00114 Positive Choline phosphate 8.5 184.07332 1014 C00588 Positive cis-4-Hydroxy-D-proline 7.6 132.06552 440014 C03440 Positive Citrulline 8.6 176.10297 9750 C00327 Positive Coenzyme A 7.75 768.12248 87642 C00010 Positive Creatine 8 132.07675 586 C00300 Positive Creatinine 4.7 114.06619 588 C00791 Positive Cystathionine 9.5 223.0747 439258 C02291 Positive Cytosine 5.45 112.05054 597 C00380 Positive DAlanine 7.9 90.05496 71080 C00133 Positive Dethiobiotin 4.6 215.13902 445027 C01909 Positive Diethanolamine 7.5 106.08626 8113 C06772 Positive Dihydrouracil 7.8 115.0502 649 C00429 Positive Glucosamine 7.88 180.08665 439213 C00329 Positive Glutamine 8.1 147.07642 5961 C00064 Positive Glutathione 8.5 308.09108 124886 C00051 Positive Glycine 8.5 76.0393 750 C00037 Positive Guanidoacetic acid 8.5 118.0611 763 C00581 Positive Homoserine 7.7 120.06552 12647 C00263 Positive Hypotaurine 8.15 110.02703 107812 C00519 Positive Hypoxanthine 5.21 137.04579 135398638 C00262 Positive Inosine 5.59 269.08805 135398641 C00294 Positive Isoleucine 5.5 132.10191 6306 C00407 Positive Kynurenine 5.4 209.09207 161166 C00328 Positive Leucine 5.3 132.10191 6106 C00123 Positive Methionine 5.7 150.05833 6137 C00073 Positive N-Acetylasparagine 6.2 175.07133 99715 0 Positive N-Acetyl-D-galactosamine 6.02 222.09721 35717 C01074 Positive N-Acetylneuraminic acid 7.5 310.11326 3568 C00270 Positive NAD+ 8 664.1164 5893 C00003 Positive NADP+ 10 744.08273 5886 C00006 Positive Niacinamide 3.33 123.05529 936 C00153 Positive N-Methylalanine 6.23 104.07061 5288725 C02721 Positive Norleucine 5.25 132.10191 21236 C01933 Positive Norvaline 5.7 118.08626 439575 C01799 Positive Ophthalmic acid 7.5 290.13466 7018721 C21016 Positive o-Succinylhomoserine 7.95 220.08156 439406 C01118  Positive Oxidized glutathione 10.5 613.15924 65359 C00127 Positive Pantothenic acid 4.5 220.11795 6613 C00864 Positive Phenylalanine 4.87 166.08626 6140 C00079 Positive Phosphoethanolamine 9.3 142.02637 1015 C00346 Positive Proline 6.76 116.07061 145742 C00148 Positive Pyridoxal 4 168.06552 1050 C00250 Positive S-Adenosylhomocysteine 7 385.12886 439155 C00021 Positive S-Adenosylmethionine 8.4 400.15234 34756 C00019 Positive Sarcosine 7.97 90.05496 1088 C00213 Positive Serine 8.5 106.04987 5951 C00716 Positive Taurine 8.1 126.02194 1123 C00245 Positive Thiamine 10 266.11958 1130 C00378 Positive Threonine 7.8 120.06552 6288 C00188 Positive trans-4-Hydroxyproline 7.6 132.06552 5810 C01157 Positive Trigonelline 5.94 138.05496 5570 C01004 Positive Tryptophan 5.9 205.09715 6305 C00078 Positive Tyrosine 6.65 182.08117 6057 C00082 Positive Valine 5.7 118.08626 6287 C00183 Positive METABOLITES_END #END