#METABOLOMICS WORKBENCH spinelli_lab_20251112_131016 DATATRACK_ID:6673 STUDY_ID:ST004394 ANALYSIS_ID:AN007343 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 20, 2025, 5:21 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 Polar Metabolomics on Lymphoblastoid cell lines (LCL) betweeen individuals with ST:STUDY_TITLE Fragile X Syndrome and from typically developing male controls. ST:STUDY_SUMMARY Extract and measure polar metabolites from Lymphoblastoid cell lines (LCL) from ST:STUDY_SUMMARY individuals with Fragile X Syndrome versus from typically developing male ST:STUDY_SUMMARY 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:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS101.raw SUBJECT_SAMPLE_FACTORS - FXS1_02 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS102.raw SUBJECT_SAMPLE_FACTORS - FXS1_03 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS103.raw SUBJECT_SAMPLE_FACTORS - FXS2_01 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS201.raw SUBJECT_SAMPLE_FACTORS - FXS2_02 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS202.raw SUBJECT_SAMPLE_FACTORS - FXS2_03 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_FXS203.raw SUBJECT_SAMPLE_FACTORS - TD_01 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_WT01.raw SUBJECT_SAMPLE_FACTORS - TD_02 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240108_RichterLab_SS_Cell_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)). 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 After the cells were pelleted, samples were incubated on dry ice with cold 80% SP:SAMPLEPREP_SUMMARY methanol. The extracts were spun down and the supernatant was vacuum dried SP:SAMPLEPREP_SUMMARY 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_01 FXS1_02 FXS1_03 FXS2_01 FXS2_02 FXS2_03 TD_01 TD_02 Factors Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS1 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:FXS2 Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD Sample source:Intracellular Male Human Lymphoblastoid Cell Lines | Genotype:TD Carnitine 122584228.3 166153976.8 95702770.9 156298017 162614731.4 114461544.9 68445914.99 67847377.84 Inosine 1343603.152 2572610.016 1103873.536 10100836.72 13255987.24 9357363.48 5019814.593 7611121.456 Phosphoethanolamine 0 0 0 1913174.752 2023417.297 1791997.333 13089082.05 11509017.88 Oxidized glutathione 114177.6383 211644.502 128654.1264 118606.3785 38020.64637 0 242328.5259 216619.8385 L-Histidinol 0 717640.8939 1010982.433 2025849.223 2676124.583 2390697.28 13242490.34 12002661.67 UDP-N-acetylgalactosamine 2889261.552 3761286.009 2639237.132 3844507.494 3614427.041 2881866.592 2995131.375 2559168.978 Trigonelline 7526644.648 11371214.23 6057471.999 20590845.32 26473178.3 20708286.17 13293339.63 22230807.86 Alanine 13429213.1 15807516.96 9796800.204 18146339.7 18738608.73 13849223.76 13273724.57 14810210.68 2-Hydroxyphenethylamine 7400219.749 11355144.23 5761718.325 20679000.82 26501840.76 20659525.5 14220003.46 22433868.78 Nicotinic acid 18129057.6 22031391.91 16703879.64 20470446.51 20325191.43 13235317.89 16465986.26 16724780.25 Lysine 1485654.964 812543.8353 1227950.968 865293.43 1158194.848 0 2461558.635 2911082.344 3-Methyladenine 11253869.37 9067322.886 7168016.941 7478157.54 7572977.18 7316257.989 7243727.578 8066757.682 Arginine 40918406.3 50563171.65 34855862.38 47539077.71 66343920.52 33075863.77 89631176.64 136108833.8 Tryptophan 7185556.02 9125837.32 6058230.011 9838239.559 8223787.446 7114920.895 9130720.603 7282815.804 Serine 255490.7988 0 372310.1345 1007540.351 1268162.778 979602.7596 2267238.627 2613389.456 NAD+ 2963989.284 3517030.02 2064804.855 1957979.427 2117381.978 1638486.609 3835670.692 3652510.203 Cys-Gly 1679458.122 2605723.389 2787819.518 1701043.495 1973092.871 1992818.053 3797669.801 2755615.245 S-Adenosylmethionine 8943549.173 14350344.51 8545346.75 12031303.18 12244576.45 6892290.854 10338921.89 10712619.54 Choline phosphate 311303975.7 418090434.9 407958795.2 441581107.8 391537548.6 268965620.1 836198821.1 706068743.4 Niacinamide 221646800.9 279925860.8 187664829.6 249820048.1 234727558.3 169450405.3 207417690.5 237954820.7 Biotin 1432515.591 1885565.584 939957.6746 1361039.767 1437254.5 697824.6022 1782276.995 2706400.685 Taurine 76401584.43 93390434.48 65961745.54 52960413.05 53683364.32 39833303.6 97304201.08 82640813.89 Diethanolamine 7638974.046 5702533.892 5151952.587 5513939.468 6151002.721 6352240.192 6214327.895 6642012.793 Phenylalanine 6096083.367 8071696.294 6400888.564 11370359.18 11691166.33 7467547.018 16009002.07 20119058.73 Glutathione 49975315.62 68373290.54 53510615.22 48132857.67 47977392.78 34523472.92 88194533.3 72113503.35 D-Ornithine 4312165.585 3373980.038 2164708.664 3024298.443 4711844.643 1735304.896 2871575.789 3506880.948 Pyridoxine 11036600.3 12960002.72 11323192.62 15013759.36 18181635.07 10389703.03 20854612.61 28427917.07 Creatine 454272155 584794633 315672067.1 457874890.9 474654098.6 314470599.2 485606162.3 467430821.8 Glutamine 230475016.1 297977472.7 237598437.5 302462710.1 314330433.4 213991904.9 463872383.7 454322251.4 Leucine 13384135.38 15936886.64 11727216.22 19329951.95 19712304.01 12375209.06 25969255.23 29686982.73 cis-4-Hydroxy-D-proline 467567484.9 603011082.8 322371222.7 463828730.2 485536287.7 323955369.4 497305749.5 482467029.4 Carnosine 1066364.317 1420085.952 962918.618 2245564.217 2460737.056 896215.1069 3509000.983 3897533.105 Asparagine 8964756.666 10645943.96 6958057.192 13915091.07 12350569.09 10250761.16 19542857.86 19155932.07 Choline 16432021.83 26978528.65 10448371.51 27687606.2 26418852.39 18728205.1 39689888.09 37809001.47 Tyrosine 3773823.699 3056650.374 2853522.257 4681626.406 4964598.222 3619610.141 6100378.522 6584759.943 Ornithine 4242503.374 4557787.327 3021105.593 2904188.509 4583825.931 1735304.896 3078570.679 4158543.434 Homoserine 3396796.94 4004416.54 3702583.577 5039440.3 4677327.234 2852652.77 6546453.487 5990506.664 Glycine 678970.2387 636458.7888 0 848919.708 948813.7143 0 1401612.434 847420.3145 1-Methylhistidine 838760.176 1446508.809 1089387.535 2057659.787 2191486.267 1238748.668 3276701.534 3130965.629 Proline 1250385900 1574900620 1056324607 1058825801 1119179485 806158249.2 1307360739 1154064983 Threonine 3442827.092 4009802.876 3702583.577 4941235.21 5369476.75 3225866.696 6580818.321 6635653.543 Methionine 4666632.027 5015968.458 4074685.67 6403980.442 8125993.185 5864344.653 9470184.547 10044618.82 Citrulline 1616518.211 1704024.097 1124347.196 1501016.364 2265442.27 1380568.887 2088272.308 2502682.302 Hypotaurine 5774822.945 7695613.892 5161913.344 4526846.34 4339258.28 3197425.902 7231929.413 5941957.491 Norvaline 217101005.4 269083367.7 203819626.9 735167710.8 743808883.1 507725434.5 854778837.3 872157593 Guanine 9229632.71 12004231.75 6664163.173 11203150 9986274.174 8892448.475 9799643.025 16320904.1 Isoleucine 14167553.16 20264156.2 13651684.4 21544681.53 25840139.93 15039142.81 27942209.11 27604746.61 Guanidoacetic acid 4541105.499 7178468.262 4684897.495 5189819.567 6131280.408 4522904.234 8389872.105 7762942.53 Valine 230181263.6 306556764.3 225142614.8 770701626.6 820314087.3 569544917.7 976095699.6 964262763.4 beta-Alanine 7829843.909 8627993.903 5903062.313 5401559.106 4599896.392 3548728.085 7512147.968 6079021.217 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity Carnitine 7 162.1125 10917 C00318 Positive Inosine 5.59 269.0881 135398641 C00294 Positive Phosphoethanolamine 9.3 142.0264 1015 C00346 Positive Oxidized glutathione 10.5 613.1592 65359 C00127 Positive L-Histidinol 9.77 142.0975 165271 C00860 Positive UDP-N-acetylgalactosamine 9 608.0889 1167 C00203 Positive Trigonelline 5.94 138.055 5570 C01004 Positive Alanine 7.9 90.055 5950 C00041 Positive 2-Hydroxyphenethylamine 5.61 138.0913 1000 C02735 Positive Nicotinic acid 4.14 124.0393 938 C00253 Positive Lysine 12.6 147.1128 5962 C00047 Positive 3-Methyladenine 4.8 150.0774 135398661 C00913 Positive Arginine 13 175.119 6322 C00062 Positive Tryptophan 5.9 205.0972 6305 C00078 Positive Serine 8.5 106.0499 5951 C00716 Positive NAD+ 8 664.1164 5893 C00003 Positive Cys-Gly 8.5 179.0485 439498 C01419 Positive S-Adenosylmethionine 8.7 400.1523 34756 C00019 Positive Choline phosphate 8.5 184.0733 1014 C00588 Positive Niacinamide 3.33 123.0553 936 C00153 Positive Biotin 4.5 245.0954 171548 C00120 Positive Taurine 8.1 126.0219 1123 C00245 Positive Diethanolamine 7.9 106.0863 8113 C06772 Positive Phenylalanine 4.87 166.0863 6140 C00079 Positive Glutathione 8.5 308.0911 124886 C00051 Positive D-Ornithine 11.5 133.0972 71082 C00515 Positive Pyridoxine 3.7 170.0812 1054 C00314 Positive Creatine 8 132.0768 586 C00300 Positive Glutamine 8.1 147.0764 5961 C00064 Positive Leucine 5.3 132.1019 6106 C00123 Positive cis-4-Hydroxy-D-proline 7.8 132.0655 440014 C03440 Positive Carnosine 8.4 227.1139 439224 C00386 Positive Asparagine 8.26 133.0608 6267 C00152 Positive Choline 9.75 105.1148 305 C00114 Positive Tyrosine 6.65 182.0812 6057 C00082 Positive Ornithine 11.5 133.0972 6262 C01602 Positive Homoserine 7.7 120.0655 12647 C00263 Positive Glycine 8.5 76.0393 750 C00037 Positive 1-Methylhistidine 9.72 170.0924 92105 C01152 Positive Proline 6.76 116.0706 145742 C00148 Positive Threonine 7.8 120.0655 6288 C00188 Positive Methionine 5.7 150.0583 6137 C00073 Positive Citrulline 8.6 176.103 9750 C00327 Positive Hypotaurine 8.15 110.027 107812 C00519 Positive Norvaline 5.7 118.0863 439575 C01799 Positive Guanine 5.7 152.0567 135398634 C00242 Positive Isoleucine 5.5 132.1019 6306 C00407 Positive Guanidoacetic acid 8.5 118.0611 763 C00581 Positive Valine 5.7 118.0863 6287 C00183 Positive beta-Alanine 7.8 90.055 239 C00099 Positive METABOLITES_END #END