#METABOLOMICS WORKBENCH spinelli_lab_20251113_085852 DATATRACK_ID:6688 STUDY_ID:ST004404 ANALYSIS_ID:AN007363 PROJECT_ID:PR002783 VERSION 1 CREATED_ON November 26, 2025, 12:55 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 L-Glutamine-13C5,15N2 stable isotope tracing on cell and media of 4-week iPSC ST:STUDY_TITLE derived forebrain excitatory neurons generated from a FXS and CRISPR edited ST:STUDY_TITLE isogenic control. ST:STUDY_SUMMARY To test whether the reduced steady-state glutamate levels in FXS neurons were ST:STUDY_SUMMARY due to impaired synthesis, we traced [¹³C₅,¹⁵N₂]-glutamine in 4-week ST:STUDY_SUMMARY induced pluripotent stem cells (iPSC) derived forebrain excitatory neurons ST:STUDY_SUMMARY generated from a Fragile X Syndrome (FXS) and CRISPR edited isogenic control. ST:STUDY_SUMMARY LC/MS polar metabolomics data showed that glutamine was not efficiently ST:STUDY_SUMMARY converted into glutamate and consequently fewer labeled carbons enter the ST:STUDY_SUMMARY oxidative and reductive TCA cycle (reflected by reduced levels of labeled α-KG, ST:STUDY_SUMMARY fumarate, malate, and aspartate). However, the percent labeled glutamate ST:STUDY_SUMMARY secreted to the media by FXS neurons was increased, suggesting altered ST:STUDY_SUMMARY neurotransmission. 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 iControl 1 Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:TD | Sample type:Cultured cells RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S21.raw SUBJECT_SAMPLE_FACTORS - cell iControl 2 Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:TD | Sample type:Cultured cells RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S22.raw SUBJECT_SAMPLE_FACTORS - cell iControl 3 Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:TD | Sample type:Cultured cells RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S23.raw SUBJECT_SAMPLE_FACTORS - cell FXS 1 Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:FXS | Sample type:Cultured cells RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S24.raw SUBJECT_SAMPLE_FACTORS - cell FXS 2 Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:FXS | Sample type:Cultured cells RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S25.raw SUBJECT_SAMPLE_FACTORS - cell FXS 3 Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:FXS | Sample type:Cultured cells RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S26.raw SUBJECT_SAMPLE_FACTORS - blank 1 Sample source:blank media | Genotype:NA | Sample type:Blank RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S27.raw SUBJECT_SAMPLE_FACTORS - blank 2 Sample source:blank media | Genotype:NA | Sample type:Blank RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S28.raw SUBJECT_SAMPLE_FACTORS - media iControl 1 Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:TD | Sample type:Culture media RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S07.raw SUBJECT_SAMPLE_FACTORS - media iControl 2 Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:TD | Sample type:Culture media RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S08.raw SUBJECT_SAMPLE_FACTORS - media iControl 3 Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:TD | Sample type:Culture media RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S09.raw SUBJECT_SAMPLE_FACTORS - media FXS 1 Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:FXS | Sample type:Culture media RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S10.raw SUBJECT_SAMPLE_FACTORS - media FXS 2 Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:FXS | Sample type:Culture media RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S11.raw SUBJECT_SAMPLE_FACTORS - media FXS 3 Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:FXS | Sample type:Culture media RAW_FILE_NAME=20250110_MK_Sneha_Neuron_Basal_Metabolomics_and_Glutamine_Tracing_S12.raw #COLLECTION CO:COLLECTION_SUMMARY iPSC lines were cultured on Matrigel coated plates in mTeSR™1 (Catalog #85850) CO:COLLECTION_SUMMARY with 10uM ROCK inhibitor (Y-27632). Neural induction was performed using the CO:COLLECTION_SUMMARY embryoid body formation protocol from StemCell technologies. Briefly cells were CO:COLLECTION_SUMMARY plated on Matrigel coated AggreWell™800 wells in STEMdiff™ Neural Induction CO:COLLECTION_SUMMARY Medium + SMADi and embryoid bodies were allowed to form for 13 days followed by CO:COLLECTION_SUMMARY Rosette selection as per manufactures instructions. The rosettes were plated on CO:COLLECTION_SUMMARY Poly-L-Ornithine (PLO)/ laminin coated plates for 3 days and then dissociated CO:COLLECTION_SUMMARY into single cells and expanded as NPCs in STEMdiff™ Neural Progenitor Medium. CO:COLLECTION_SUMMARY To generate neuron precursors, NPCs were cultured in STEMdiff™ Forebrain CO:COLLECTION_SUMMARY Neuron Differentiation media (Catalog #08600) for 3 days on PLO/laminin plates CO:COLLECTION_SUMMARY followed by replating at a density of 135,000 cells/cm2 in PLO/laminin plates in CO:COLLECTION_SUMMARY STEMdiff™ Forebrain Neuron Maturation media (Catalog #08605) with Compound E CO:COLLECTION_SUMMARY for 4 weeks. CO:SAMPLE_TYPE Culture Cells and Extracellular Media #TREATMENT TR:TREATMENT_SUMMARY For glutamine flux tracing experiments, cells were cultured for 8hrs in TR:TREATMENT_SUMMARY glutamine free media supplemented with 2mM L-Glutamine-13C5,15N2 TR:TREATMENT_SUMMARY (Catalog#607983, Millipore Sigma). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For extraction from cell 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. For SP:SAMPLEPREP_SUMMARY media, 10ul of sample was added to 90ul of 80% methanol and vortexed for 10mins SP:SAMPLEPREP_SUMMARY before spinning to collect the supernatant containing extracted metabolites and SP:SAMPLEPREP_SUMMARY vacuum dried. The extract was then 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 cell iControl 1 cell iControl 2 cell iControl 3 cell FXS 1 cell FXS 2 cell FXS 3 media iControl 1 media iControl 2 media iControl 3 media FXS 1 media FXS 2 media FXS 3 blank 1 blank 2 Factors Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:TD | Sample type:Cultured cells Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:TD | Sample type:Cultured cells Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:TD | Sample type:Cultured cells Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:FXS | Sample type:Cultured cells Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:FXS | Sample type:Cultured cells Sample source:Male Homo sapien iPSCs derived neurons cell | Genotype:FXS | Sample type:Cultured cells Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:TD | Sample type:Culture media Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:TD | Sample type:Culture media Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:TD | Sample type:Culture media Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:FXS | Sample type:Culture media Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:FXS | Sample type:Culture media Sample source:Male Homo sapien iPSCs derived neurons media | Genotype:FXS | Sample type:Culture media Sample source:blank media | Genotype:NA | Sample type:Blank Sample source:blank media | Genotype:NA | Sample type:Blank Glutamine 241887329 219762532 180522318 420180895.1 402340170.9 289542444.2 89510674.3 73683544.62 82796374.47 92931409.16 76534691.52 55285482.05 53658931.81 30061421.72 Glutamine_13C1 34599692.88 30438399.13 24922185.76 55887136.32 55899231.37 38667362.35 9447587.548 8838081.699 9270192.35 9848165.613 7227018.498 4997159.247 1409886.366 471742.7215 Glutamine_13C2 17603632.33 16103448.37 12199432.65 27853345.66 29717581.9 16585466.11 5489190.069 4081485.56 3849566.982 2325919.713 3078686.543 745200.4736 451480.0811 0 Glutamine_13C3 29672111.56 24882717.22 21445933.92 57756744.16 57481354.04 41387297.18 8997306.447 7644576.024 8694820.488 9794338.679 7783184.074 6513282.205 0 0 Glutamine_13C4 2769855.768 3551256.17 3275091.827 5937474.355 6395863.16 4611815.777 0 497522.8782 2460096.024 2076919.028 100925.2839 0 495316.055 0 Glutamine_13C5 67887817.39 61541233.4 45750410.99 124400135.8 118757998.3 94174973.4 21625591.49 18936900.82 19823740.42 25373390.02 20714306.48 18718940.56 0 0 Glutamine_15N1 162484325.8 145623876.8 117217356.9 438564110.5 408490748.8 298659023.6 42450843.6 35206324.09 38800336.33 51797304.28 45780158.9 32908699.79 0 0 Glutamine_15N2 46056571.56 43356073.87 37090891.89 116421768.5 109795087.8 77815453.3 15199489.82 11087416.57 13468947.34 17455259.13 16702158.16 13531607.81 9072545.652 6098695.046 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention index Quantified m/z PubChem ID KEGG ID Polarity Glutamine 8.1 147.07642 5961 C00064 Positive Glutamine_13C1 8.1 148.07977 5961 C00064 Positive Glutamine_13C2 8.1 149.08313 5961 C00064 Positive Glutamine_13C3 8.1 150.08648 5961 C00064 Positive Glutamine_13C4 8.1 151.08984 5961 C00064 Positive Glutamine_13C5 8.1 152.09319 5961 C00064 Positive Glutamine_15N1 8.1 148.07345 5961 C00064 Positive Glutamine_15N2 8.1 149.07049 5961 C00064 Positive METABOLITES_END #END