#METABOLOMICS WORKBENCH silviaradenkovic_20231219_094045 DATATRACK_ID:4538 STUDY_ID:ST003019 ANALYSIS_ID:AN004953 PROJECT_ID:PR001759 VERSION 1 CREATED_ON December 19, 2023, 2:43 pm #PROJECT PR:PROJECT_TITLE Metabolomic profiling of PMM2-CDG brain organoids part 3 PR:PROJECT_SUMMARY Summary PMM2-CDG is a rare inborn error of metabolism caused by deficiency of PR:PROJECT_SUMMARY the PMM enzyme, which leads to impaired protein glycosylation. While the PR:PROJECT_SUMMARY disorder has primarily neurological presentation, there is limited knowledge PR:PROJECT_SUMMARY about the specific brain-related changes that result from PMM deficiency. PR:PROJECT_SUMMARY Utilizing 3D brain organoids derived from individuals with PMM2-CDG, we PR:PROJECT_SUMMARY identified abnormal glucose metabolism in PMM2-CDG organoids, indicating PR:PROJECT_SUMMARY disturbances in metabolic utilization. In this experiment, day 40 organoids were PR:PROJECT_SUMMARY used and GC/MS was performed to specifically quantify sugars such as polyols and PR:PROJECT_SUMMARY hexoses. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Radenkovic PR:FIRST_NAME Silvia PR:ADDRESS 200 2nd Ave SW Rochester MN PR:EMAIL radenkovic.silvia@mayo.edu PR:PHONE 507(77) 6-6107 PR:FUNDING_SOURCE NIH, KU Leuven #STUDY ST:STUDY_TITLE Metabolomic profiling of PMM2-CDG brain organoids by GC/MS ST:STUDY_SUMMARY PMM2-CDG is a rare inborn error of metabolism caused by deficiency of the PMM ST:STUDY_SUMMARY enzyme, which leads to impaired protein glycosylation. While the disorder has ST:STUDY_SUMMARY primarily neurological presentation, there is limited knowledge about the ST:STUDY_SUMMARY specific brain-related changes that result from PMM deficiency. Utilizing 3D ST:STUDY_SUMMARY brain organoids derived from individuals with PMM2-CDG, we identified abnormal ST:STUDY_SUMMARY glucose metabolism in PMM2-CDG organoids, indicating disturbances in metabolic ST:STUDY_SUMMARY utilization. In this experiment, day 160 organoids were used. ST:INSTITUTE Mayo Clinic ST:LAST_NAME Radenkovic ST:FIRST_NAME Silvia ST:ADDRESS 200 2nd Ave SW Rochester MN, USA ST:EMAIL radenkovic.silvia@mayo.edu ST:PHONE 507(77) 6-6107 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN WT/PMM2-CDG SU:AGE_OR_AGE_RANGE 5-25 SU:GENDER Male and female #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 C1 S1 Genotype:CTR RAW_FILE_NAME=SR01 SUBJECT_SAMPLE_FACTORS P1 S2 Genotype:PMM2 RAW_FILE_NAME=SR02 SUBJECT_SAMPLE_FACTORS C2 S3 Genotype:CTR RAW_FILE_NAME=SR03 SUBJECT_SAMPLE_FACTORS P2 S4 Genotype:PMM2 RAW_FILE_NAME=SR04 SUBJECT_SAMPLE_FACTORS C3 S5 Genotype:CTR RAW_FILE_NAME=SR05 SUBJECT_SAMPLE_FACTORS P3 S6 Genotype:PMM2 RAW_FILE_NAME=SR06 #COLLECTION CO:COLLECTION_SUMMARY Brain were collected at day in vitro 160. Briefly, 5 organoids per cell line CO:COLLECTION_SUMMARY were collected and washed three times in DPBS (Gibco). The DPBS was then CO:COLLECTION_SUMMARY removed, the organoids flash frozen and kept in -80 °C prior to metabolomics CO:COLLECTION_SUMMARY experiments. The metabolites were extracted using two phase extraction protocol. CO:COLLECTION_SUMMARY First, the organoids were transferred to lysing matrix tube and 350 µL of CO:COLLECTION_SUMMARY ice-cold extraction buffer (80 % MeOH, IS) was added to the sample. Next, the CO:COLLECTION_SUMMARY organoids were lyzed with ribolyzer, the lysate transferred to 1.5 mL Eppendorf CO:COLLECTION_SUMMARY tube and placed overnight at -80 °C. Further, the samples were centrifuged at CO:COLLECTION_SUMMARY 15,000 rpm, 4 °C, 20 min. 100 µL of supernatant was transferred to a fresh CO:COLLECTION_SUMMARY Eppendorf tube and 35 µL of ddH20 was added, followed by 800 µL 100% CO:COLLECTION_SUMMARY chloroform. The samples were then vortexed and stored at 4 °C overnight. Next, CO:COLLECTION_SUMMARY the polar phase was the used for Gas Chromatography/Mass Spectrometry (GC/MS) CO:SAMPLE_TYPE Brain organoids CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Samples were not treated #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The metabolites were extracted from the brain organoids using double phase SP:SAMPLEPREP_SUMMARY metabolite extration. Standards containing metabolites of interest were prepared SP:SAMPLEPREP_SUMMARY simultaneously and dried overnight together with cellular extracts by vacuum SP:SAMPLEPREP_SUMMARY centrifugation at 4 °C. The following day, 20 µL of methoxyamine (MOX) (Sigma SP:SAMPLEPREP_SUMMARY Aldrich) was added to the samples and they were incubated for 90 min at 37 °C. SP:SAMPLEPREP_SUMMARY Next, 60 µL of N, O-bis(trimethylsilyl)trifluoroacetamide (TMS) (Sigma Aldrich) SP:SAMPLEPREP_SUMMARY was added to the samples, which were then incubated at 60 °C for 30 min. SP:SAMPLEPREP_SUMMARY Samples were kept overnight in a dry and cool place to allow further SP:SAMPLEPREP_SUMMARY derivatization with TMS SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_DERIVATIZATION MOX, TMS #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Agilent 7890A GC (Agilent Technologies) coupled with an HP-5 ms 5 % phenyl CH:CHROMATOGRAPHY_SUMMARY methyl silox capillary column (30 m, 0.25 mm, 0.25 um, Agilent Technologies) CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent HP-5ms (30m x 0.25mm, 0.25um) CH:SOLVENT_A N/A CH:SOLVENT_B N/A CH:FLOW_GRADIENT The temperature gradient applied was as follows: 2 min 100°C, next 175 °C at CH:FLOW_GRADIENT Δ20 °C/min, followed by an increase to 230 °C at Δ4 °C/min, kept at 230 °C CH:FLOW_GRADIENT for 3 min, increased to 300 °C at Δ 40°C/min and lastly kept at 300 °C for 5 CH:FLOW_GRADIENT min. The column was further baked for another 3 min at 325 °C after the CH:FLOW_GRADIENT gradient. CH:FLOW_RATE NA CH:COLUMN_TEMPERATURE Gradient #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7000B MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE EI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Mass Hunter Workstation software with the Quantitative Analysis Version MS:MS_COMMENTS B.06.00/Build 6.0.388.0 Specific metabolites were identified based on their MS:MS_COMMENTS fragment formula (SIM)/elution time #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC MS_METABOLITE_DATA_START Samples S1 S2 S3 S4 S5 S6 Factors Genotype:CTR Genotype:PMM2 Genotype:CTR Genotype:PMM2 Genotype:CTR Genotype:PMM2 Fructose_oxTMS_307_abundant 1.315087642 1.09158405 0.868233333 0.741163641 0.816679025 0.710954379 Galactose_oxTMS 1.00055359 3.107120254 0.729658974 3.040358416 1.269787436 1.738929599 Glucose_oxTMS 1.065007976 0.871516994 0.871790376 0.589548098 1.063201648 0.992997595 Mannitol_oxTMS 1.092587181 0.74129157 0.924007145 0.746709126 0.983405674 0.783476124 Sorbitol_oXTMS_307 1.00583954 0.808770683 1.022566337 0.578601147 0.971594123 0.681856687 Galactitol_oxTMS_307 1.390668617 1.008691416 0.667865773 0.834605051 0.94146561 0.706650726 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Fructose_oxTMS_307_abundant Galactose_oxTMS Glucose_oxTMS Mannitol_oxTMS Sorbitol_oXTMS_307 Galactitol_oxTMS_307 METABOLITES_END #END