#METABOLOMICS WORKBENCH silviaradenkovic_20240306_124721 DATATRACK_ID:4689 STUDY_ID:ST003121 ANALYSIS_ID:AN005115 PROJECT_ID:PR001939 VERSION 1 CREATED_ON March 12, 2024, 2:18 pm #PROJECT PR:PROJECT_TITLE Metabolomic profiling of ALG13-CDG brain organoids PR:PROJECT_SUMMARY ALG13-CDG is a rare inborn error of metabolism caused by deficiency of the ALG13 PR:PROJECT_SUMMARY enzyme, which is believed to lead 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 ALG13 deficiency. We PR:PROJECT_SUMMARY used 3D brain organoids derived from fibroblasts of the individuals with PR:PROJECT_SUMMARY ALG13-CDG to probe the brain-specific metabolic changes in ALG13-CDG. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Radenkovic PR:FIRST_NAME Silvia PR:ADDRESS 200 2nd Ave SW Rochester MN PR:EMAIL silradenkovic@gmail.com PR:PHONE 507(77) 6-6107 PR:FUNDING_SOURCE NIH #STUDY ST:STUDY_TITLE Metabolomic profiling of ALG13-CDG brain organoids ST:STUDY_SUMMARY ALG13-CDG is a rare inborn error of metabolism caused by deficiency of the ALG13 ST:STUDY_SUMMARY enzyme, which is believed to lead to impaired protein glycosylation. While the ST:STUDY_SUMMARY disorder has primarily neurological presentation, there is limited knowledge ST:STUDY_SUMMARY about the specific brain-related changes that result from ALG13 deficiency. We ST:STUDY_SUMMARY used 3D brain organoids derived from fibroblasts of the individuals with ST:STUDY_SUMMARY ALG13-CDG to probe the brain-specific metabolic changes in ALG13-CDG. ST:INSTITUTE Mayo Clinic ST:LAST_NAME Radenkovic ST:FIRST_NAME Silvia ST:ADDRESS 200 2nd Ave SW Rochester MN, USA ST:EMAIL silradenkovic@gmail.com ST:PHONE 507(77) 6-6107 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN WT/ALG13-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 11207 SR01 Genotype:ALG13 | Sample source:Brain organoids RAW_FILE_NAME=SR01; DAYS IN VITRO=77; Weight in mg=17 SUBJECT_SAMPLE_FACTORS 11740 SR02 Genotype:ALG13 | Sample source:Brain organoids RAW_FILE_NAME=SR02; DAYS IN VITRO=77; Weight in mg=23 SUBJECT_SAMPLE_FACTORS 11816 SR03 Genotype:ALG13 | Sample source:Brain organoids RAW_FILE_NAME=SR03; DAYS IN VITRO=77; Weight in mg=21 SUBJECT_SAMPLE_FACTORS 5381 SR04 Genotype:CTR | Sample source:Brain organoids RAW_FILE_NAME=SR04; DAYS IN VITRO=77; Weight in mg=5 SUBJECT_SAMPLE_FACTORS 5400 SR05 Genotype:CTR | Sample source:Brain organoids RAW_FILE_NAME=SR05; DAYS IN VITRO=77; Weight in mg=25 SUBJECT_SAMPLE_FACTORS 8856.3 SR06 Genotype:CTR | Sample source:Brain organoids RAW_FILE_NAME=SR06; DAYS IN VITRO=77; Weight in mg=13 #COLLECTION CO:COLLECTION_SUMMARY Brain were collected at day in vitro 77. CO:SAMPLE_TYPE Brain organoids CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY NA #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, 4-5 organoids per cell line were collected and washed three times in SP:SAMPLEPREP_SUMMARY PBS. The PBS was then removed, the organoids flash frozen and kept in -80 °C SP:SAMPLEPREP_SUMMARY prior to metabolomics experiments. The metabolites were extracted using two SP:SAMPLEPREP_SUMMARY phase extraction protocol. First, the organoids were transferred to lysing SP:SAMPLEPREP_SUMMARY matrix tube and 350 µL of ice-cold extraction buffer (80 % MeOH, IS) was added SP:SAMPLEPREP_SUMMARY to the sample. Next, the organoids were lyzed with ribolyzer, the lysate SP:SAMPLEPREP_SUMMARY transferred to 1.5 mL Eppendorf tube and placed overnight at -80 °C. Further, SP:SAMPLEPREP_SUMMARY the samples were centrifuged at 15,000 rpm, 4 °C, 20 min. 100 µL of SP:SAMPLEPREP_SUMMARY supernatant was transferred to a fresh Eppendorf tube and 35 µL of ddH20 was SP:SAMPLEPREP_SUMMARY added, followed by 800 µL 100% chloroform. The samples were then vortexed and SP:SAMPLEPREP_SUMMARY stored at 4 °C overnight. Next, the polar phase was then used for Liquid SP:SAMPLEPREP_SUMMARY Chromatography/Mass Spectrometry (LC/MS). SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY C18 iP REVERSE PHASE CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 10mM tributylamine; 15mM acetic acid CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B CH:FLOW_GRADIENT until 2 min post injection. A linear gradient to 37% B was carried out until 7 CH:FLOW_GRADIENT min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient CH:FLOW_GRADIENT increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the CH:FLOW_GRADIENT gradient returned to 5% B. The chromatography was stopped at 40 min CH:FLOW_RATE 0.25 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS El-Maven polly, ThermoFisher Xcalibur, Metabolites were annotated based on the MS:MS_COMMENTS in-house metabolite library- elution time and m/z values #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC MS_METABOLITE_DATA_START Samples SR01 SR02 SR03 SR04 SR05 SR06 Factors Genotype:ALG13 | Sample source:Brain organoids Genotype:ALG13 | Sample source:Brain organoids Genotype:ALG13 | Sample source:Brain organoids Genotype:CTR | Sample source:Brain organoids Genotype:CTR | Sample source:Brain organoids Genotype:CTR | Sample source:Brain organoids 13C5-D5-15N Glutamic acid 360E+06 302E+06 301E+06 506E+06 298E+06 388E+06 PEP 265E+06 203E+06 238E+06 512E+05 340E+06 102E+06 L-Lactic_acid 108E+09 115E+09 121E+09 540E+08 126E+09 107E+09 Hexose-P 188E+07 256E+07 319E+07 814E+06 276E+07 265E+07 3PG 182E+07 235E+07 244E+07 744E+06 327E+07 745E+06 Hexose 103E+08 112E+08 101E+08 589E+07 151E+08 691E+07 GA3P 460E+06 708E+06 707E+06 143E+06 957E+06 441E+06 DHAP 645E+07 711E+07 652E+07 271E+07 917E+07 440E+07 Hexose-bisphosphate 106E+07 875E+06 180E+07 398E+06 106E+07 577E+06 Hexosamine-P 676E+05 740E+05 934E+05 133E+05 615E+05 619E+05 Ribitol 665E+05 992E+05 943E+05 444E+05 110E+06 805E+05 Sorbitol 317E+07 327E+07 440E+07 139E+07 458E+07 370E+07 Fumarate 149E+07 147E+07 161E+07 752E+06 149E+07 147E+07 Citrate 585E+07 618E+07 642E+07 235E+07 768E+07 411E+07 Malate 310E+08 337E+08 347E+08 178E+08 319E+08 331E+08 Succinate 162E+08 223E+08 225E+08 425E+07 163E+08 163E+08 L-Glutamic acid 151E+08 154E+08 171E+08 373E+07 174E+08 132E+08 L-Glutamine 410E+08 411E+08 512E+08 157E+08 507E+08 357E+08 L-Methionine 127E+07 144E+07 172E+07 368E+06 192E+07 103E+07 L-Phenylalanine 591E+07 633E+07 752E+07 136E+07 871E+07 437E+07 Glycine 105E+07 901E+06 134E+07 418E+06 724E+06 106E+07 L-Alanine 232E+07 235E+07 287E+07 108E+07 275E+07 205E+07 L-Histidine 478E+06 423E+06 441E+06 222E+06 620E+06 358E+06 L-Proline 908E+06 972E+06 107E+07 628E+06 845E+06 709E+06 L-Serine 240E+07 225E+07 318E+07 798E+06 241E+07 187E+07 L-Tryptophan 282E+06 320E+06 312E+06 205E+06 368E+06 265E+06 L-Arginine 232E+06 265E+06 320E+06 134E+06 322E+06 212E+06 L-Asparagine 480E+06 466E+06 668E+06 317E+06 592E+06 517E+06 L-Isoleucine 139E+08 155E+08 186E+08 380E+07 199E+08 105E+08 L-Threonine 805E+07 852E+07 830E+07 287E+07 100E+08 593E+07 L-Cysteine 150E+05 107E+05 146E+05 182E+05 162E+05 141E+05 L-Lysine 381E+06 370E+06 438E+06 207E+06 522E+06 306E+06 L-Aspartic acid 468E+07 477E+07 549E+07 107E+07 417E+07 364E+07 L-Tyrosine 334E+07 377E+07 446E+07 105E+07 473E+07 239E+07 Taurine 152E+07 145E+07 339E+07 693E+06 369E+07 402E+07 ADP 145E+07 145E+07 203E+07 660E+06 250E+07 121E+07 AMP 126E+07 161E+07 259E+07 993E+06 256E+07 222E+07 ATP 538E+06 316E+06 489E+06 171E+06 467E+06 260E+06 CDP 319E+05 297E+05 374E+05 257E+05 518E+05 199E+05 CMP 110E+06 102E+06 152E+06 189E+06 180E+06 147E+06 CTP 149E+05 701E+04 138E+05 548E+04 132E+05 710E+04 GDP 298E+05 244E+05 506E+05 253E+05 502E+05 199E+05 GMP 119E+06 140E+06 269E+06 964E+05 245E+06 186E+06 GTP 805E+04 438E+04 105E+05 502E+04 733E+04 475E+04 TDP 243E+05 283E+05 282E+05 158E+04 245E+05 238E+05 TMP 261E+05 154E+05 180E+05 122E+05 373E+05 202E+05 UDP 267E+06 249E+06 325E+06 137E+06 465E+06 205E+06 UMP 903E+06 109E+07 148E+07 612E+06 182E+07 148E+07 UTP 749E+05 430E+05 697E+05 222E+05 645E+05 370E+05 Cysteineglutathione disulfide 237E+06 342E+06 408E+06 140E+05 449E+06 176E+06 Glutathione 293E+08 305E+08 312E+08 139E+08 321E+08 279E+08 NAD 261E+07 270E+07 288E+07 741E+06 273E+07 263E+07 NADH 124E+06 159E+06 153E+06 202E+05 119E+06 109E+06 NADP 207E+05 154E+05 257E+05 398E+04 269E+05 110E+05 NADPH 178E+05 946E+04 200E+05 425E+04 209E+05 917E+04 Oxidized_glutathione 166E+06 623E+06 639E+06 157E+05 729E+06 240E+06 GDP-D-Mannose 242E+06 203E+06 297E+06 788E+05 243E+06 203E+06 GDP-L-Fucose 126E+06 820E+05 115E+06 296E+05 118E+06 103E+06 UDP-Hexose 116E+07 114E+07 119E+07 329E+06 133E+07 133E+07 UDP-glucuronate 555E+06 289E+06 368E+06 115E+06 575E+06 382E+06 UDP-GlcNAc 453E+07 424E+07 437E+07 113E+07 382E+07 434E+07 6PG 492E+05 520E+05 597E+05 155E+05 825E+05 231E+05 Erythrose-P 616E+05 459E+05 631E+05 173E+05 613E+05 445E+05 Pentose 195E+06 243E+06 214E+06 110E+06 289E+06 146E+06 D-Ribose_5-phosphate 161E+07 198E+07 236E+07 557E+06 207E+07 148E+07 D-Ribulose_5-phosphate 215E+06 199E+06 246E+06 673E+05 228E+06 244E+06 D-Sedoheptulose 113E+05 793E+04 107E+05 636E+04 166E+05 168E+05 Sedoheptulose-P 822E+06 910E+06 108E+07 160E+06 796E+06 823E+06 G3P 227E+08 369E+08 312E+08 832E+07 361E+08 218E+08 Xylulose 5-phosphate 273E+06 263E+06 411E+06 174E+06 277E+06 256E+06 Acetyl-CoA 104E+05 105E+05 160E+05 736E+03 131E+05 342E+04 Coenzyme_A 186E+04 765E+03 412E+04 000E+00 104E+04 124E+04 Phosphocreatine 264E+06 342E+06 471E+06 479E+05 296E+06 244E+06 2-Ketobutyric_acid 582E+06 635E+06 538E+06 311E+06 872E+06 387E+06 Betaine 661E+07 723E+07 839E+07 227E+07 847E+07 562E+07 CMP-sialic acid 422E+06 524E+06 751E+06 211E+05 614E+06 424E+06 Cyclic AMP 185E+05 225E+05 519E+05 793E+04 211E+05 421E+05 Cytidine 115E+05 916E+04 532E+04 236E+04 714E+04 875E+04 FAD 221E+05 211E+05 374E+05 756E+04 337E+05 230E+05 GABA 839E+06 847E+06 962E+06 233E+06 909E+06 752E+06 Hypoxanthine 688E+05 342E+05 477E+05 187E+05 473E+05 824E+05 IMP 178E+06 142E+06 143E+06 246E+05 211E+06 178E+06 Inosine 712E+04 265E+04 653E+04 386E+04 533E+04 182E+05 N-Acetyl-D-hexosamine 125E+05 103E+05 152E+05 240E+04 557E+04 278E+04 N-Acetyl-L-aspartate 580E+08 602E+08 662E+08 175E+08 558E+08 488E+08 N-Acetylneuraminic acid 240E+07 280E+07 555E+07 150E+07 420E+07 245E+07 Orotic acid 160E+06 205E+06 140E+06 417E+05 104E+06 194E+06 Thymidine 238E+05 219E+05 402E+05 145E+05 293E+05 991E+04 Uridine 266E+04 696E+04 145E+05 599E+04 535E+04 106E+05 Xanthine 539E+05 649E+05 106E+06 159E+05 132E+06 294E+05 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB 13C5-D5-15N Glutamic acid NA PEP HMDB00263 L-Lactic_acid HMDB00190 Hexose-P HMDB01401 3PG HMDB00807 Hexose HMDB00122 GA3P HMDB01112 DHAP HMDB01473 Hexose-bisphosphate HMDB01058 Hexosamine-P HMDB01254 Ribitol HMDB0000508 Sorbitol HMDB00247 Fumarate HMDB00134 Citrate HMDB00094 Malate HMDB00156 Succinate HMDB00254 L-Glutamic acid HMDB00148 L-Glutamine HMDB00641 L-Methionine HMDB00696 L-Phenylalanine HMDB00159 Glycine HMDB00123 L-Alanine HMDB00161 L-Histidine HMDB00177 L-Proline HMDB00162 L-Serine HMDB00187 L-Tryptophan HMDB00929 L-Arginine HMDB00517 L-Asparagine HMDB00168 L-Isoleucine HMDB00172 L-Threonine HMDB00167 L-Cysteine HMDB00574 L-Lysine HMDB00182 L-Aspartic acid HMDB0000191 L-Tyrosine HMDB0000158 Taurine HMDB00251 ADP HMDB01341 AMP HMDB00045 ATP HMDB00538 CDP HMDB01546 CMP HMDB00095 CTP HMDB00082 GDP HMDB01201 GMP HMDB01397 GTP HMDB01273 TDP HMDB01274 TMP HMDB01227 UDP HMDB00295 UMP HMDB00288 UTP HMDB00285 Cysteineglutathione disulfide HMDB0000656 Glutathione HMDB00125 NAD HMDB00902 NADH HMDB01487 NADP HMDB00217 NADPH HMDB00221 Oxidized_glutathione HMDB03337 GDP-D-Mannose HMDB01163 GDP-L-Fucose HMDB01095 UDP-Hexose HMDB00286 UDP-glucuronate HMDB00935 UDP-GlcNAc HMDB00290 6PG HMDB01316 Erythrose-P HMDB01321 Pentose HMDB0000283 D-Ribose_5-phosphate HMDB01548 D-Ribulose_5-phosphate HMDB00618 D-Sedoheptulose HMDB0003219 Sedoheptulose-P HMDB01068 G3P HMDB00126 Xylulose 5-phosphate HMDB00868 Acetyl-CoA HMDB01206 Coenzyme_A HMDB01423 Phosphocreatine HMDB0001511 2-Ketobutyric_acid HMDB00005 Betaine HMDB00043 CMP-sialic acid HMDB0001176 Cyclic AMP HMDB0000058 Cytidine HMDB0000089 FAD HMDB01248 GABA HMDB00112 Hypoxanthine HMDB0000157 IMP HMDB0000175 Inosine HMDB0000195 N-Acetyl-D-hexosamine HMDB00215 N-Acetyl-L-aspartate HMDB00812 N-Acetylneuraminic acid HMDB00230 Orotic acid HMDB0000226 Thymidine HMDB0000273 Uridine HMDB0000296 Xanthine HMDB0000292 METABOLITES_END #END