#METABOLOMICS WORKBENCH spinelli_lab_20251112_132543 DATATRACK_ID:6674 STUDY_ID:ST004395 ANALYSIS_ID:AN007345 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 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_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 Aminoisobutyric acid 86227200.5 82266840.63 102881175.7 109909878.8 81030237.7 77224294.69 113604894.1 91899878.97 109420089.4 3-Methoxytyramine 6606331.52 9603722.383 7883541.252 6396461.904 13460817.92 5727867.119 13597663.76 7804802.478 8107371.624 3-Methylhistidine 189774995 192906581.3 199994148.3 249994842.4 201506025.4 176720352.9 306180664.7 250800184.7 284577304.8 3-Methyladenine 82695875.8 100826067.1 112441843.7 108813859.3 93110139.88 85026774.38 59476690.83 48362902.07 50616345.48 3-Methylhistamine 6579404.13 9008595.632 11190709.04 9529123.836 17985881.1 11392395.62 14541722.36 8970862.164 6696585.106 4-Acetamidobutanoic acid 29167787.6 27301782.82 28767838.51 34504274.31 27333779.48 23737055.15 30557881.12 31133820.39 34150980.35 Arginine 3628836019 3731674879 3795148117 4222782198 3707480482 3469142148 4558432664 4069810575 4542064456 Asparagine 124326543 134206947.7 129531821.4 172989715.1 118436741.1 112319212.2 195048636 163825025.7 179577337.8 beta-Alanine 6941855.23 6130027.903 7244648.776 8664331.005 8578417.103 3867134.043 5618539.976 5989510.081 6262081.924 Biotin 47473554.5 49741750.45 47793063.49 57721584.32 48089350.1 46573955.96 54181144.42 52735912.76 58495795.27 Carnosine 165780458 169250364.4 172569489.2 198528175.4 170485335.2 152345681 193284040.6 177008573.1 191490760.1 Choline 315955604 306914002.1 310176299.9 358253830.9 298920257.9 284761460.8 418543958.1 399755015.3 428139876.4 cis-4-Hydroxy-D-proline 5681458310 5582596772 5719758104 6969217808 5753347370 5093260366 7263530148 6683769057 7078775145 Citrulline 80332738.4 83124580.68 87574955.23 111174155.2 90506518.07 79460609.04 106251758.1 92147469.01 96980881.87 Creatine 5681849063 5583845469 5728734694 6969637668 5752245149 5089654412 7268402705 6683875442 7076424712 Cys-Gly 0 3527390.892 0 1870409.339 0 1789747.941 0 2417420.896 4647981.067 Cytosine 230275387 203901436.2 228754277.4 515015464.8 434010752.6 386104807.1 68235976.89 55489802.31 63111203.5 2-Amino-2-deoxy-D-mannose 8035605.28 7506476.362 6710960.981 9788318.073 8276668.262 6659137.282 10047960.22 7967246.461 9347690.309 D-Ornithine 477628104 480984299.3 508808458.4 603164724.5 489150477.5 480457480.2 666959603.1 607851513 624365769.5 Diethanolamine 21810526.5 18110008.28 19906601.21 17390580.76 19842440.76 22664380.81 18340613.9 21276772.71 20738485.16 Galactosamine 11784784 12356972.62 12073219.76 9733857.999 8304269.442 6716614.941 10046347.11 8946013.067 9360421.39 Glucosamine 8122054.56 7644170.887 6675439.498 9688585.501 8183091.166 6574918.669 10036936.13 7922644.548 9405445.966 Glutamine 4185313754 3966194702 3936251266 4267167013 3418182288 3019700728 6591349819 6137805163 6391796272 CAR DC5:0 5891291.63 5557266.33 6228715.941 6332323.968 4713509.787 4594377.902 6341199.197 5709051.417 6376505.634 Glycine 4159954.62 5324115.193 4791081.867 7033519.606 5288618.509 5367619.662 5017523.964 4036646.657 6531997.975 Guanidoacetic acid 70580932.7 99622833.53 104667322.3 94289162.23 80731369.31 79202336.39 37509366.89 38054399.51 36552982.3 Guanine 975135.565 1690089.561 1218123.532 0 0 0 0 0 0 Homoserine 68106029.8 62598945.06 57538809.69 105702931 73995969.85 68111862.98 205355903.4 181527838 200797700.8 Hypotaurine 141830067 130529553.9 138671554.1 203090149.5 148305638.3 126406443.5 254161137.5 240745128.8 288305215 Hypoxanthine 37574952.1 47157413.75 51255303.39 50226688.37 38355379.23 35776583.21 5297052.274 4406821.992 5502496.067 Isoleucine 1626312246 1561310973 1540201129 1861703781 1414110432 1337473786 2272515661 1993398917 2344172469 Kynurenine 9926376.45 9477511.753 9637407.623 10629112.43 9144572.017 8674893.93 13361413.42 11406152.35 12546354.5 Alanine 174258876 176020014.2 186949777.6 268858021.1 227926542.5 198383310.6 201215716 180070997.2 196670686.4 Carnitine 757030830 750674595.1 785431683.5 875676771.4 717442169.5 663452336.6 875983303 813777126.4 919532514.4 Ornithine 477634874 480984299.3 508812137.3 603164724.5 489164469.9 480457480.2 666998715.3 607881334.6 624365769.5 Leucine 891940519 797692467.5 847029458.6 1082092016 887257877.8 760042212.3 1660218374 1596987292 1767114730 Lysine 232572678 208647068.2 214145152 332326754.2 261042594.8 241400500.9 589859985.1 527680856.1 557987087.3 Methionine 305417463 287119536.1 272733427.7 383223809.3 299127083.1 272896394 557928303.5 502476967.7 564944451.9 N-Acetylneuraminic acid 7293193.17 6178020.59 6464455.72 10266358.84 8427138.73 6643648.091 14372178.13 19288821.43 22902530.07 N-Methylalanine 85922719.9 91323114.97 94783963.87 103206994.9 83261890.96 82486504.88 105468137 98123409.84 108940342.9 Niacinamide 404306295 410842120.7 424758889.8 488768550.4 360544883.8 351686561.3 473110976.2 466524076.2 509425977.7 Nicotinic acid 28253077.2 27060534 28633239.65 35447876.77 26777172.89 24886141.54 32958176.43 30695780.91 33807010.83 Norvaline 6785024475 7026989420 7305305082 7581179452 6567900670 6202108252 7650517957 6943723325 7830952890 Phenylalanine 430091453 403531772.6 396490615.6 572486873.9 433836645.2 399577089.6 804540235.7 766573463.6 861136698.7 2-Hydroxyphenethylamine 0 0 0 0 0 0 31535049.48 32819465.94 28444917.28 Choline phosphate 46995317.1 56233696.41 59765228.32 42693809.98 37860381.51 27282739.4 25842677.44 17701212.6 17070889.01 Proline 6656708931 7286449409 7655072515 7000504398 5966556029 5657041397 5582014626 5143782158 5701029338 Pyridoxal 48758962.2 61046879.31 65014300.47 66208648.78 53560346.58 56503063.87 19165935.69 18122664.63 26359440.29 Pyridoxine 1123383402 1067410440 1056556037 1343396258 1007684358 988847787.6 1458238137 1399891807 1634625663 S-Adenosylmethionine 970471.229 1832752.247 2256379.261 1437055.728 1420243.302 877544.256 397137.2218 103918.9677 223212.3529 Serine 14551766.7 8320321.406 8917369.867 21959176.97 16624017.95 15873414.65 60853693.05 54142298.21 62462803.01 Taurine 11534196.9 13385858.22 12489592.36 19005861.51 12583990.6 10988416.14 16559919.79 13140503.94 18840462.21 Threonine 68106029.8 55563201.38 57538809.69 97730059.8 70685358.74 60131237.24 205355903.4 181818387.5 200797700.8 Thymine 4472876.36 5324418.799 5322574.279 6140032.476 5722482.478 5346142.163 6575331.437 6387100.325 7077754.128 Trigonelline 3248593.1 0 0 0 0 0 31042378.77 32562389.19 28513632.72 Tryptophan 84297139.9 79114303.89 75081119.68 98495845.42 77199507.84 73029392.32 178247575.6 153063771.9 181834440.9 Tyrosine 141596927 126698790 131428977.9 167403753.5 132413273.8 126862442.7 269741059.3 238539283.3 264055323.2 Valine 6784164838 7026989420 7305305082 7581179452 6567900670 6200504255 7650517957 6943723325 7830952890 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity Aminoisobutyric acid 8.4 104.0706 6119 C03665 Positive 3-Methoxytyramine 8.03 168.10191 1669 C05587 Positive 3-Methylhistidine 6.6 170.0924 64969 C01152 Positive 3-Methyladenine 4.8 150.0774 135398661 C00913 Positive 3-Methylhistamine 7.5 126.10257 69520 0 Positive 4-Acetamidobutanoic acid 4.7 146.0812 18189 C02946 Positive Arginine 13 175.119 6322 C00062 Positive Asparagine 8.26 133.0608 6267 C00152 Positive beta-Alanine 7.8 90.055 239 C00099 Positive Biotin 4.5 245.0954 171548 C00120 Positive Carnosine 8.4 227.1139 439224 C00386 Positive Choline 9.75 105.1148 305 C00114 Positive cis-4-Hydroxy-D-proline 7.8 132.0655 440014 C03440 Positive Citrulline 8.6 176.103 9750 C00327 Positive Creatine 8 132.0768 586 C00300 Positive Cys-Gly 8.5 179.0485 439498 C01419 Positive Cytosine 6 112.0505 597 C00380 Positive 2-Amino-2-deoxy-D-mannose 7.51 180.0867 440049 C03570 Positive D-Ornithine 11.5 133.0972 71082 C00515 Positive Diethanolamine 7.9 106.0863 8113 C06772 Positive Galactosamine 8.21 180.0867 3034366 0 Positive Glucosamine 7.5 180.0867 439213 C00329 Positive Glutamine 8.1 147.0764 5961 C00064 Positive CAR DC5:0 7.17 276.1442 71317118 0 Positive Glycine 8.5 76.0393 750 C00037 Positive Guanidoacetic acid 8.5 118.0611 763 C00581 Positive Guanine 5.7 152.0567 135398634 C00242 Positive Homoserine 7.7 120.0655 12647 C00263 Positive Hypotaurine 8.15 110.027 107812 C00519 Positive Hypoxanthine 5.21 137.0458 135398638 C00262 Positive Isoleucine 5.5 132.1019 6306 C00407 Positive Kynurenine 5.4 209.0921 161166 C00328 Positive Alanine 7.9 90.055 71080 C00133 Positive Carnitine 7 162.1125 10917 C00318 Positive Ornithine 11.5 133.0972 6262 C01602 Positive Leucine 5.3 132.1019 6106 C00123 Positive Lysine 12.6 147.1128 5962 C00047 Positive Methionine 5.7 150.0583 6137 C00073 Positive N-Acetylneuraminic acid 7.5 310.1133 3568 C00270 Positive N-Methylalanine 6.23 104.0706 5288725 C02721 Positive Niacinamide 3.33 123.0553 936 C00153 Positive Nicotinic acid 4.14 124.0393 938 C00253 Positive Norvaline 5.7 118.0863 439575 C01799 Positive Phenylalanine 4.87 166.0863 6140 C00079 Positive 2-Hydroxyphenethylamine 5.61 138.0913 1000 C02735 Positive Choline phosphate 8.5 184.0733 1014 C00588 Positive Proline 6.76 116.0706 145742 C00148 Positive Pyridoxal 4 168.0655 1050 C00250 Positive Pyridoxine 3.7 170.0812 1054 C00314 Positive S-Adenosylmethionine 8.7 400.1523 34756 C00019 Positive Serine 8.5 106.0499 5951 C00716 Positive Taurine 8.1 126.0219 1123 C00245 Positive Threonine 7.8 120.0655 6288 C00188 Positive Thymine 3.3 127.0502 1135 C00178 Positive Trigonelline 5.94 138.055 5570 C01004 Positive Tryptophan 5.9 205.0972 6305 C00078 Positive Tyrosine 6.65 182.0812 6057 C00082 Positive Valine 5.7 118.0863 6287 C00183 Positive METABOLITES_END #END