#METABOLOMICS WORKBENCH spinelli_lab_20251112_125321 DATATRACK_ID:6671 STUDY_ID:ST004393 ANALYSIS_ID:AN007342 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 20, 2025, 5:12 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 metabolites in human male FXS plasma vs TD controls ST:STUDY_SUMMARY Polar metabolites were measured and compared from plasma of human male with FXS ST:STUDY_SUMMARY (N=6) versus age-matched TD 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 Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male #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 - FXS02 Sample source:Homo Sapien Male Plasma | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_FXS02.raw SUBJECT_SAMPLE_FACTORS - FXS03 Sample source:Homo Sapien Male Plasma | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_FXS03.raw SUBJECT_SAMPLE_FACTORS - FXS04 Sample source:Homo Sapien Male Plasma | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_FXS04.raw SUBJECT_SAMPLE_FACTORS - FXS05 Sample source:Homo Sapien Male Plasma | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_FXS05.raw SUBJECT_SAMPLE_FACTORS - FXS06 Sample source:Homo Sapien Male Plasma | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_FXS06.raw SUBJECT_SAMPLE_FACTORS - TD01 Sample source:Homo Sapien Male Plasma | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_TD01.raw SUBJECT_SAMPLE_FACTORS - TD02 Sample source:Homo Sapien Male Plasma | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_TD02.raw SUBJECT_SAMPLE_FACTORS - TD03 Sample source:Homo Sapien Male Plasma | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_TD03.raw SUBJECT_SAMPLE_FACTORS - TD04 Sample source:Homo Sapien Male Plasma | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_TD04.raw SUBJECT_SAMPLE_FACTORS - TD05 Sample source:Homo Sapien Male Plasma | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_TD05.raw SUBJECT_SAMPLE_FACTORS - TD06 Sample source:Homo Sapien Male Plasma | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_TD06.raw SUBJECT_SAMPLE_FACTORS - TD07 Sample source:Homo Sapien Male Plasma | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240816_SnehaCellMediaSerum_TD07.raw #COLLECTION CO:COLLECTION_SUMMARY FXS plasma samples were collected at Rush University Medical Center and TD CO:COLLECTION_SUMMARY plasma samples collected at UMASS Chan Medical School. Fresh blood samples were CO:COLLECTION_SUMMARY collected in purple cap K2EDTA tubes and spun at 3000 RPM for 10 minutes at 4deg CO:COLLECTION_SUMMARY to separate the plasma. All participants or their legal guardians, as CO:COLLECTION_SUMMARY appropriate, signed informed consent to the study. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY No special treatment. Tissue samples were collected from patient. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For plasma, 10ul of sample was added to 90ul of 80% methanol and vortexed for SP:SAMPLEPREP_SUMMARY 10mins before spinning to collect the supernatant containing extracted SP:SAMPLEPREP_SUMMARY metabolites and vacuum dried. The extract was then resuspended in LC/MS grade SP:SAMPLEPREP_SUMMARY 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 FXS02 FXS03 FXS04 FXS05 FXS06 TD01 TD02 TD03 TD04 TD05 TD06 TD07 Factors Sample source:Homo Sapien Male Plasma | Genotype:FXS Sample source:Homo Sapien Male Plasma | Genotype:FXS Sample source:Homo Sapien Male Plasma | Genotype:FXS Sample source:Homo Sapien Male Plasma | Genotype:FXS Sample source:Homo Sapien Male Plasma | Genotype:FXS Sample source:Homo Sapien Male Plasma | Genotype:TD Sample source:Homo Sapien Male Plasma | Genotype:TD Sample source:Homo Sapien Male Plasma | Genotype:TD Sample source:Homo Sapien Male Plasma | Genotype:TD Sample source:Homo Sapien Male Plasma | Genotype:TD Sample source:Homo Sapien Male Plasma | Genotype:TD Sample source:Homo Sapien Male Plasma | Genotype:TD 3-(2-Hydroxyphenyl)propanoic acid 348741.8219 146562.8854 105488.4632 267898.5338 269582.4295 162949.7469 151043.7849 210192.1431 241023.8733 207139.7137 326140.0693 286241.4536 3E-Methylglutaconic acid 1704786.486 837929.5097 1839167.118 671063.936 300216.4017 6393493.872 0 4372735.626 636454.8049 2203562.136 427700.1408 191137.65 Adenosine 355857.9484 326391.7336 550366.4259 371150.0855 739143.2124 1171358.547 1640509.446 1332795.742 1052109.299 1201473.698 1166379.445 765880.8541 ADP-ribose 540526.926 571268.6672 858565.3915 621901.5885 1016615.42 1740097.173 2421394.954 1920925.028 1591486.821 1365253.002 1745705.619 1131936.152 ATP 82938.0189 75041.51581 171926.701 103668.3364 197714.3301 276263.0196 495317.7531 388934.2224 317763.5263 250255.6086 283616.2186 213460.2066 Aspartic acid 3072495.195 3160849.798 3539354.434 6140865.237 2613771.702 14235168.77 3169708.538 9593481.351 4182219.512 2736466.644 2529977.003 3367869.047 Cellobiose 49823995.63 46404758.76 41899577.92 43490772.22 37088413.56 32583839.5 38677934.47 23154819.66 32003316.71 25578464.85 34529055.67 32209332.21 Cortisol 177914.7795 75428.9089 87633.76301 104169.0773 117259.7848 144783.9015 73747.93769 70458.3669 86357.21209 112124.2125 97825.03495 121130.1299 3',5' cyclic AMP 25692.96881 14341.24898 21025.08283 14257.01113 9659.693149 9939.822871 14907.49755 7638.009614 9369.503455 27673.96707 31674.8898 19226.65274 Ribose 5-phosphate 0 1557.686484 0 2157.717755 0 14479.37503 16015.57408 16569.7461 24628.91859 3302.28453 4526.160096 13910.79372 dAMP 0 0 0 0 0 0 1756.292497 14866.56543 9878.353159 12714.46669 18689.96805 18113.42819 dUTP 0 1969.57274 0 0 0 0 4821.937207 12394.36827 33823.65909 31250.58938 21586.46665 33718.73212 Fructose 6-phosphate 9596.609678 5250.686494 20871.18147 15135.99151 17176.49987 35368.37795 13411.62081 0 14288.8029 9907.972675 55494.20461 16043.8157 Fumaric acid 0 0 126707.3999 113227.5957 0 524703.019 765285.8811 132367.1635 850124.0135 1099188.213 327283.2075 608704.2051 Gluconic acid 0 0 0 0 0 5547990.522 0 4358385.4 14454928.03 18920504.75 35476941.92 40420260.14 Glucosamine 6-phosphate 0 1353.592776 0 4229.059049 0 6270.925621 0 0 7386.24183 15434.99026 12448.12596 10969.58417 Glucose 1-phosphate 9596.609678 5250.686494 20871.18147 15135.99151 17176.49987 35368.37795 10771.78453 0 14288.8029 9907.972675 55494.20461 16043.8157 Glucose 6-phosphate 20491.99862 17831.77915 20391.76149 31322.85444 22171.83963 21648.8742 33299.31154 14368.2232 43420.87284 33411.05978 31937.82815 19571.97335 Glutamic acid 967910.1002 8188541.54 7018289.405 1727810.848 12992125.37 12028878.64 11064064.51 6706442.545 24440890.43 8489794.233 6305379.177 10688184.15 Glycolic acid 257366.9839 202773.7927 132510.8676 2346446.273 906825.0375 1001490.428 464098.9653 725041.0219 1010903.619 489779.8434 365964.8623 1679706.793 Guanosine 44986.86286 44948.82397 53118.83758 37613.21846 61076.47963 78865.3822 92662.24454 74664.16114 76554.67496 84320.65692 68782.36718 62399.6428 GDP 0 3871.049881 10200.9883 7404.520208 20183.14393 53228.16426 59120.73434 52898.833 42888.87964 48216.15494 48542.177 24081.53025 GMP 9214.15881 22624.70968 29338.45301 11666.82233 43868.14048 72969.53442 108485.2085 89430.31229 67733.13113 66074.29689 77287.45998 22017.7938 Homovanillic acid 0 6859181.951 3549922.347 8675406.145 4567090.455 8290106.845 10865099.09 6049450.154 9421947.769 8180484.398 16475163.36 8659552.579 Hydroxyphenyllactic acid 0 6859181.951 3549922.347 8675406.145 4567090.455 8290106.845 10865099.09 6049450.154 9421947.769 8180484.398 16475163.36 8659552.579 Indoxyl sulfate 83630803.14 106940930.8 53782218.73 92834804.73 42059016.67 4263167.445 7320634.503 15588816.19 15403661.32 48252350.24 35810255.4 9883080.845 IDP 0 0 0 0 0 0 6325.009963 58750.81756 330888.1719 1501954.467 741614.3291 721289.4639 IMP 15962.46536 37880.82409 124270.3441 32734.57283 76011.37902 236992.2169 250201.229 173858.0229 77303.46147 151287.1635 162394.9143 165098.3186 Lactose 46246476.6 39104126.24 33948630.21 38135266.22 28376561.27 24776585.71 29885647.15 21711345.17 26591799.93 21841428.99 33083440.72 26050454.92 2-Oxoglutaric acid 79445.75984 135559.5816 801823.6319 0 397163.3682 3760831.878 5191241.482 5965703.792 8923702.576 8039966.492 8095972.827 9785333.268 Palatinose 46246476.6 39248760.75 33948630.21 38135266.22 28203775.31 25037969.54 29654681.44 21884936.31 26591799.93 22121504.57 31123920.59 23085423.08 Pregnenolone sulfate 735732.3003 415793.6788 728582.7717 1437311.659 917718.8752 1053281.235 390151.4226 874375.7088 535012.7567 1092814.867 709180.7925 1709360.217 Glucaric acid 39811.43499 0 126707.3999 113227.5957 0 524703.019 765285.8811 132367.1635 850124.0135 1099188.213 327283.2075 608704.2051 Stachyose 32795.57262 64142.52743 71879.7232 69808.4932 199636.4919 178943.8221 343841.9633 252191.7796 389974.9999 179068.4962 234237.842 119375.9495 Trehalose 46246476.6 37863442.05 33948630.21 37967536.79 28376561.27 25233185.54 29885647.15 21884936.31 26591799.93 22041389.42 31973872.31 25543546.28 Uridine 2905038.363 4993962 4139532.372 2327459.664 4466813.828 5359796.007 3897098.202 3173067.716 4802981.662 2728283.854 3182180.651 5145301.805 UDP 3610.074466 0 3337.993507 1986.164853 12959.1044 16452.16462 44391.65211 38082.76665 11036.15311 2115.691681 2350.059471 0 UDP-glucose 7459.891966 18205.25542 17211.97788 6260.220542 31313.15185 44615.13278 128034.5139 34670.63671 35264.77416 27767.73103 9532.732252 0 UDP-glucuronic acid 0 0 2342.560097 0 11145.02187 6945.609854 43216.97338 10413.83886 17209.41851 33971.03942 29341.81166 17334.75015 UDP-N-acetylglucosamine 0 62905.7708 16420.07511 28864.49037 96536.54838 88428.87787 179915.359 102894.9277 146189.7088 125190.6963 116536.8683 100024.143 UDP-galactose 7459.891966 18205.25542 17211.97788 6260.220542 31313.15185 44615.13278 128034.5139 34670.63671 35264.77416 27767.73103 9532.732252 0 UMP 0 9918.471328 0 7019.810739 13544.39865 26631.35034 49261.30515 30130.99454 30156.6064 23268.43841 56144.64968 20865.48581 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity 3-(2-Hydroxyphenyl)propanoic acid 2.67 165.0557 873 C01198 Negative 3E-Methylglutaconic acid 8 143.035 1551553 0 Negative Adenosine 4.4 266.0895 60961 C00212 Negative ADP-ribose 8.7 558.0644 33576 C00301 Negative ATP 10 505.9885 5957 C00002 Negative Aspartic acid 8.6 132.0302 5960 C00049 Negative Cellobiose 8.3 341.1089 10712 C06422 Negative Cortisol 2.38 361.2021 657311 C00735 Negative 3',5' cyclic AMP 5 328.0452 6076 C00575 Negative Ribose 5-phosphate 9.49 229.0119 440101 C00117 Negative dAMP 7.2 330.0609 12599 C00360 Negative dUTP 10.91 466.9663 65070 C00460 Negative Fructose 6-phosphate 9.5 259.0224 69507 C00085 Negative Fumaric acid 9.5 115.0037 444972 C00122 Negative Gluconic acid 7.8 195.051 10690 C00257 Negative Glucosamine 6-phosphate 9 258.0384 439217 C00352 Negative Glucose 1-phosphate 9.5 259.0224 65533 C00103 Negative Glucose 6-phosphate 10 259.0224 5958 C00092 Negative Glutamic acid 8.5 146.0459 33032 C00025 Negative Glycolic acid 3.31 75.0088 757 C00160 Negative Guanosine 6.5 282.0844 135398635 C00387 Negative GDP 10.9 442.0171 135398619 C00035 Negative GMP 9.9 362.0507 135398631 C00144 Negative Homovanillic acid 4.74 181.0506 1738 C05582 Negative Hydroxyphenyllactic acid 4.7 181.0506 9378 C03672 Negative Indoxyl sulfate 4.18 212.0023 10258 0 Negative IDP 10.87 427.0062 135398651 C00104 Negative IMP 9.3 347.0398 135398640 C00130 Negative Lactose 8.3 341.1089 6134 C01970 Negative 2-Oxoglutaric acid 9.3 145.0143 51 C00026 Negative Palatinose 8.5 341.1089 439559 C017424 Negative Pregnenolone sulfate 2.13 395.1898 20845972 C18044 Negative Glucaric acid 10 209.0303 33037 C00818 Negative Stachyose 10.05 665.2146 439531 C01613 Negative Trehalose 8.3 341.1089 7427 C01083 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-glucuronic acid 11.8 579.027 17473 C00167 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 METABOLITES_END #END