#METABOLOMICS WORKBENCH spinelli_lab_20251112_193657 DATATRACK_ID:6677 STUDY_ID:ST004397 ANALYSIS_ID:AN007349 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 26, 2025, 11:56 am #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 [¹³C₅,¹⁵N₂]-glutamine stable isotope tracing in TD, FXS2 (partial ST:STUDY_TITLE FMRP), and FXS1 (no FMRP) from Lymphoblastoid cell lines (LCL) ST:STUDY_SUMMARY Fragile X mental retardation protein (FMRP) regulates glutamine catabolism. To ST:STUDY_SUMMARY investigate Gln utilization in Fragile X Syndrome (FXS) patient-derived ST:STUDY_SUMMARY lymphoblast cell lines (LCLs), [¹³C₅,¹⁵N₂]-glutamine stable isotope ST:STUDY_SUMMARY tracing was performed in TD, FXS2 (partial FMRP), and FXS1 (no FMRP) from ST:STUDY_SUMMARY Lymphoblastoid cell lines (LCL) for 8 hours and collect polar metabolites for ST:STUDY_SUMMARY analysis. These data demonstrate the sufficiency for FMRP splicing to alter ST:STUDY_SUMMARY glutamine breakdown. 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 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 - cell WT4 1 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C01 SUBJECT_SAMPLE_FACTORS - cell WT4 2 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C02 SUBJECT_SAMPLE_FACTORS - cell WT4 3 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C03 SUBJECT_SAMPLE_FACTORS - cell FXS1 1 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C04 SUBJECT_SAMPLE_FACTORS - cell FXS1 2 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C05 SUBJECT_SAMPLE_FACTORS - cell FXS1 3 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C06 SUBJECT_SAMPLE_FACTORS - cell FXS2 1 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C07 SUBJECT_SAMPLE_FACTORS - cell FXS2 2 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C08 SUBJECT_SAMPLE_FACTORS - cell FXS2 3 Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_C09 SUBJECT_SAMPLE_FACTORS - Blank Media Sample source:Cell culture lines media | Genotype:media blank with 13C515N2-gln RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_blankmedia SUBJECT_SAMPLE_FACTORS - media WT4 1 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M01 SUBJECT_SAMPLE_FACTORS - media WT4 2 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M02 SUBJECT_SAMPLE_FACTORS - media WT4 3 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:TD RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M03 SUBJECT_SAMPLE_FACTORS - media FXS1 1 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M04 SUBJECT_SAMPLE_FACTORS - media FXS1 2 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M05 SUBJECT_SAMPLE_FACTORS - media FXS1 3 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M06 SUBJECT_SAMPLE_FACTORS - media FXS2 1 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M07 SUBJECT_SAMPLE_FACTORS - media FXS2 2 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M08 SUBJECT_SAMPLE_FACTORS - media FXS2 3 Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS RAW_FILE_NAME(Raw_file_name)=20240419_RichterLab_SS_M09 #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, Culture media #TREATMENT TR:TREATMENT_SUMMARY Cells were cultured in glutamine free RPMI 1640 medium (Sigma-Aldrich) TR:TREATMENT_SUMMARY supplemented with 15% fetal bovine serum (FBS) and 2mM L-Glutamine-13C5,15N2 TR:TREATMENT_SUMMARY (Catalog#607983, Millipore Sigma) for 8 hours, maintained at 37°C with 5% CO₂ TR:TREATMENT_SUMMARY in T25 flasks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For extraction from ccell pellets from in-vitro cultures, samples were incubated SP:SAMPLEPREP_SUMMARY on dry ice with cold 80% methanol. The extracts were spun down and the SP:SAMPLEPREP_SUMMARY supernatant 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 cell WT4 1 cell WT4 2 cell WT4 3 cell FXS1 1 cell FXS1 2 cell FXS1 3 cell FXS2 1 cell FXS2 2 cell FXS2 3 Blank Media media WT4 1 media WT4 2 media WT4 3 media FXS1 1 media FXS1 2 media FXS1 3 media FXS2 1 media FXS2 2 media FXS2 3 Factors Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD Sample source:Male Homo sapien Lymphoblastoid cell lines | Genotype:TD 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:FXS 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:FXS Sample source:Cell culture lines media | Genotype:media blank with 13C515N2-gln Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:TD Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:TD Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:TD Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS Sample source:Male Homo sapien Lymphoblastoid cell lines media | Genotype:FXS Glutamine 100326938 113613346.5 118377626.4 97832401.78 98934397.16 96047308.01 71986444 78455378.37 89712588.62 134775054.5 228004844.3 268746853.9 208605164.1 180385392.2 171799960.6 166015233.5 179293519.8 173328841.7 164888265.2 Glutamine_13C1_15N2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Glutamine_13C2_15N2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Glutamine_13C3_15N2 4450771.476 5612976.644 5132322.902 6069899.425 6205927.341 5849708.639 5411865.293 5892968.941 5809126.966 14518835.89 12887851.07 14079625.46 11903908.14 12172336.31 10704381.16 8818877.71 10390543 9971796.73 9718416.287 Glutamine_13C4_15N2 81961874.99 97174785.42 101361202.1 84130990.68 88259218.86 86999569.48 62060444.92 65971386.22 73064142.6 214789694.2 216197888.3 271223269.5 205110106.7 192764259.2 182341053.6 187107449.8 180725115.6 183852296.9 162854229.9 Glutamine_13C5_15N2 1720309649 1966491094 2054949130 1756723362 1758475270 1728610231 1208459135 1291631275 1478728460 4073051485 4412961701 5431049920 4130249309 3764393884 3552531083 3552985323 3662964264 3597856955 3313314303 Proline 1032718364 1056136896 1224690506 1024753690 1074312737 982957178.4 699896695.8 756394771.4 881908461.5 1300302715 2456324461 3098448231 2281434809 2047624115 1997355260 1865818220 2031052424 2037351188 1755430204 Proline_13C1_15N1 0 0 0 0 0 0 0 0 0 Proline_13C2_15N1 144771540.8 128154782 127017932.4 186039410.8 202120846.4 186122448.5 77420561.71 81284382.17 90137929.48 0 23577929.21 32613365.57 24901263.03 31017050.26 29372216.56 29141948.55 22864402.03 20547436.85 21539160.11 Proline_13C3_15N1 122233004.2 105862422.4 102908024.7 143014769.4 155921556.9 144724964.6 79575305.76 78596415.01 91300695.59 0 6070232.882 6911497.632 4805175.508 9066715.09 10809111.7 8867495.47 5995134.922 4558508.996 3691854.085 Proline_13C4_15N1 361020524.4 318058895.7 307120146.4 534287664.3 580988708.2 539884864.7 281283803.8 277445262.2 324564677 0 35067920.26 39770982.25 27422290.11 54797569.75 61406935.1 52678546.72 38385628.19 35393006.99 31481933.07 Proline_13C5_15N1 379025831.3 329760168.7 325914516 447443133.4 487276486.7 453294841 346951338.5 344871673.8 391690332.9 43942121.28 42784528.03 54051186.72 38821072.4 63002946.68 60601297.02 80725562.63 68446264.37 50987901.52 44973205.29 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Polarity Glutamine 8.1 147.0764 5961 C00064 Positive Glutamine_13C1_15N2 8.1 149.07049 5961 C00064 Positive Glutamine_13C2_15N2 8.1 149.07049 5961 C00064 Positive Glutamine_13C3_15N2 8.1 149.07049 5961 C00064 Positive Glutamine_13C4_15N2 8.1 149.07049 5961 C00064 Positive Glutamine_13C5_15N2 8.1 149.07049 5961 C00064 Positive Proline 6.76 116.0706 145742 C00148 Positive Proline_13C1_15N1 6.76 116.0706 145742 C00148 Positive Proline_13C2_15N1 6.76 116.0706 145742 C00148 Positive Proline_13C3_15N1 6.76 116.0706 145742 C00148 Positive Proline_13C4_15N1 6.76 116.0706 145742 C00148 Positive Proline_13C5_15N1 6.76 116.0706 145742 C00148 Positive METABOLITES_END #END