#METABOLOMICS WORKBENCH araskind_20170714_124310 DATATRACK_ID:1137 STUDY_ID:ST000821 ANALYSIS_ID:AN001304 PROJECT_ID:PR000585 VERSION 1 CREATED_ON August 2, 2017, 9:48 am #PROJECT PR:PROJECT_TITLE Stimulated Raman Immunohistochemistry PR:PROJECT_TYPE MS analysis PR:PROJECT_SUMMARY Profiling of WT, IDH1 R132H, WT + 2HG, Mut + AG!5198 PR:INSTITUTE University of Michigan PR:DEPARTMENT Neurosurgery PR:LABORATORY Orringer Lab PR:LAST_NAME Orringer PR:FIRST_NAME Daniel PR:ADDRESS Ann Arbor, MI PR:EMAIL dorringe@umich.edu PR:PHONE 734-647-9222 #STUDY ST:STUDY_TITLE Metabolomic profiling of IDH1 mutation ST:STUDY_TYPE MS analysis ST:STUDY_SUMMARY We are interested in using both spontaneous and stimulated Raman microscopy to ST:STUDY_SUMMARY visualize these metabolomic changes as spectral alterations. We have two ST:STUDY_SUMMARY isogneic cell lines of normal human astrocytes differing only by a point ST:STUDY_SUMMARY mutation in the IDH-1 gene. We will work with the metabolomics core to elucidate ST:STUDY_SUMMARY the changes in central metabolism and lipid synthesys in an effort to determine ST:STUDY_SUMMARY the precise biochemical alterations underlying observed spectral differences. We ST:STUDY_SUMMARY wil then use a selective inhibitor of the IDH1 R132H to demonstrate to attempt ST:STUDY_SUMMARY to return TCA metabolome and lipidome to WT phenotype. Lastly, we will use a ST:STUDY_SUMMARY cell-permeablized variant of 2HG (2R-octyl-alpha-hydroxyglutarate) to ST:STUDY_SUMMARY recaptitulate the R132H mutant phenotype in wild-type cells, providing strong ST:STUDY_SUMMARY evidence that 2HG accumulation uderlies the metabolomic (and thus, spectral) ST:STUDY_SUMMARY changes observed. ST:INSTITUTE University of Michigan ST:DEPARTMENT Biomedical Research Core Facilities ST:LABORATORY Metabolomics core ST:LAST_NAME Kachman ST:FIRST_NAME Maureen ST:ADDRESS 6300 Brehm Tower, 1000 Wall Street, Ann Arbor, MI 48105-5714 ST:EMAIL mkachman@med.umich.edu ST:PHONE (734) 232-8175 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 3 ST:STUDY_COMMENTS The IDH-1 R132H mutation in low grade gliomas preceptitates oncogenesis through ST:STUDY_COMMENTS a neomorphic enzyme function: the reduction of alpha-ketoglutarate (aKG) to ST:STUDY_COMMENTS 2-hydroxygutarate (2HG). 2HG accumukates to extremely high (~100mM) ST:STUDY_COMMENTS concentrations in IDH1 mutant cells and has substantial, predictable metabolic ST:STUDY_COMMENTS effects, including inhibition of a-KG dependent dioxygenases and ST:STUDY_COMMENTS hypermethylation of histones and chromatin. #SUBJECT SU:SUBJECT_TYPE HUMAN SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS SU0016656 S00026530 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016657 S00026531 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016658 S00026532 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016656 S00026533 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016657 S00026534 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016658 S00026535 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016656 S00026536 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016657 S00026537 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016658 S00026538 IDH1_MUTATION_STATUS:WT SUBJECT_SAMPLE_FACTORS SU0016656 S00026539 IDH1_MUTATION_STATUS:R132H Mutant SUBJECT_SAMPLE_FACTORS SU0016657 S00026540 IDH1_MUTATION_STATUS:R132H Mutant SUBJECT_SAMPLE_FACTORS SU0016658 S00026541 IDH1_MUTATION_STATUS:R132H Mutant SUBJECT_SAMPLE_FACTORS SU0016656 S00026542 IDH1_MUTATION_STATUS:R132H Mutant SUBJECT_SAMPLE_FACTORS SU0016657 S00026543 IDH1_MUTATION_STATUS:R132H Mutant SUBJECT_SAMPLE_FACTORS SU0016658 S00026544 IDH1_MUTATION_STATUS:R132H Mutant SUBJECT_SAMPLE_FACTORS SU0016656 S00026545 IDH1_MUTATION_STATUS:R132H Mutant SUBJECT_SAMPLE_FACTORS SU0016657 S00026546 IDH1_MUTATION_STATUS:R132H Mutant SUBJECT_SAMPLE_FACTORS SU0016658 S00026547 IDH1_MUTATION_STATUS:R132H Mutant #COLLECTION CO:COLLECTION_SUMMARY - #TREATMENT TR:TREATMENT_SUMMARY - #SAMPLEPREP SP:SAMPLEPREP_SUMMARY - SP:SAMPLEPREP_PROTOCOL_FILENAME A037-2HG_cells_updated-20160721.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent CH:COLUMN_NAME Waters Acquity HSS T3 (50 x 2.1mm, 1.8um) #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE A037-2HG cells updated-20160721.pdf #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6490A QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS uM/ng protein MS_METABOLITE_DATA_START Samples S00026530 S00026531 S00026532 S00026533 S00026534 S00026535 S00026536 S00026537 S00026538 S00026539 S00026540 S00026541 S00026542 S00026543 S00026544 S00026545 S00026546 S00026547 Factors IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:WT IDH1_MUTATION_STATUS:R132H Mutant IDH1_MUTATION_STATUS:R132H Mutant IDH1_MUTATION_STATUS:R132H Mutant IDH1_MUTATION_STATUS:R132H Mutant IDH1_MUTATION_STATUS:R132H Mutant IDH1_MUTATION_STATUS:R132H Mutant IDH1_MUTATION_STATUS:R132H Mutant IDH1_MUTATION_STATUS:R132H Mutant IDH1_MUTATION_STATUS:R132H Mutant (2R)-2-HYDROXYPENTANEDIOIC ACID 4.071306714 3.098099464 4.703858735 4.798879275 3.086003529 4.044164366 2.321686412 2.332264382 2.737507717 43.54915156 38.63892668 24.66584537 38.18522914 42.39913148 41.66765007 34.76693065 33.5109968 32.91367874 (2S)-2-HYDROXYPENTANEDIOIC ACID 7.295459461 5.89119898 5.925238318 6.187509603 6.209511497 5.454204664 5.148028685 5.1653177 5.473715581 6.299511709 4.89508229 3.822237079 4.885550348 6.014301899 4.834213193 4.660787068 4.835995756 4.299657892 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name moverz_quant ri ri_type pubchem_id inchi_key kegg_id other_id other_id_type (2R)-2-HYDROXYPENTANEDIOIC ACID 439391 (2S)-2-HYDROXYPENTANEDIOIC ACID 439939 METABOLITES_END #END