#METABOLOMICS WORKBENCH amitch_20161011_130158 DATATRACK_ID:753 STUDY_ID:ST000499 ANALYSIS_ID:AN000767 PROJECT_ID:PR000376 VERSION 1 CREATED_ON October 31, 2016, 10:53 am #PROJECT PR:PROJECT_TITLE The role of GBA1 in Parkinson's disease PR:PROJECT_SUMMARY The role of GBA1 in Parkinson's disease PR:INSTITUTE German Center for Neurodegenerative Diseases PR:DEPARTMENT Mitochondria and Inflammation in Neurodegeneration PR:LAST_NAME Deleidi PR:FIRST_NAME Michela PR:ADDRESS Otfried-Müller. Str 23, 72076, Tübingen, Germany PR:EMAIL michela.deleidi@dzne.de PR:PHONE none PR:FUNDING_SOURCE DFG #STUDY ST:STUDY_TITLE NAD+ in neurodegeneration ST:STUDY_TYPE Baseline comparison of NAD metabolism in iPSC-derived neurons ST:STUDY_SUMMARY Differentiated iPSCs from patients and controls and knockout cells ST:INSTITUTE University of Florida ST:DEPARTMENT SECIM ST:LAST_NAME Deleidi ST:FIRST_NAME Michela ST:ADDRESS Otfried-Müller. Str 23, 72076, Tübingen, Germany ST:EMAIL michela.deleidi@dzne.de ST:PHONE none ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 17 #SUBJECT SU:SUBJECT_TYPE Cells 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 105 PD1 iPSC_01 Genotype:WT/N370S Gender=male SUBJECT_SAMPLE_FACTORS 105 PD1 GC iPSC_02 Genotype:WT/WT Gender=male SUBJECT_SAMPLE_FACTORS 105 PD1 KO1 iPSC_03 Genotype:KO/KO Gender=male SUBJECT_SAMPLE_FACTORS 106 PD1 iPSC_04 Genotype:WT/N370S Gender=male SUBJECT_SAMPLE_FACTORS 106 PD1 GC iPSC_05 Genotype:WT/WT Gender=male SUBJECT_SAMPLE_FACTORS 107 PD1 iPSC_06 Genotype:WT/N370S Gender=male SUBJECT_SAMPLE_FACTORS 107 PD1 GC iPSC_07 Genotype:WT/WT Gender=male SUBJECT_SAMPLE_FACTORS 107 PD1 KO1 iPSC_08 Genotype:KO/KO Gender=male SUBJECT_SAMPLE_FACTORS 107 PD1 KO2 iPSC_09 Genotype:KO/KO Gender=male SUBJECT_SAMPLE_FACTORS 108 PD1 iPSC_10 Genotype:WT/N370S Gender=male SUBJECT_SAMPLE_FACTORS 108 PD1 GC iPSC_11 Genotype:WT/WT Gender=male SUBJECT_SAMPLE_FACTORS 109 PD1 iPSC_12 Genotype:WT/N370S Gender=male SUBJECT_SAMPLE_FACTORS 109 PD1 GC iPSC_13 Genotype:WT/WT Gender=male SUBJECT_SAMPLE_FACTORS 110 PD2 iPSC_14 Genotype:WT/L444P Gender=male SUBJECT_SAMPLE_FACTORS 110 PD2 GC iPSC_15 Genotype:WT/WT Gender=male SUBJECT_SAMPLE_FACTORS 111 PD2 iPSC_16 Genotype:WT/L444P Gender=male SUBJECT_SAMPLE_FACTORS 111 PD2 GC iPSC_17 Genotype:WT/WT Gender=male #COLLECTION CO:COLLECTION_SUMMARY Cells were washed once with cold PBS, scraped off the plate in 1 ml cold PBS, CO:COLLECTION_SUMMARY transfered in pre-cooled 50 ml falcons and pelleted by centrifugation 3000rpm at CO:COLLECTION_SUMMARY 4°C. PBS was taken off and pellets were then snap frozen in liquid N2 and CO:COLLECTION_SUMMARY stored at -80°C. #TREATMENT TR:TREATMENT_SUMMARY 80% confluence, 48 hrs after last media change, differentiation medium TR:TREATMENT_SUMMARY (Neurobasal/B27/L-glut containing medium supplemented with BDNF, ascorbic acid , TR:TREATMENT_SUMMARY GDNF, TGFβ3, dibutyryl cAMP and DAPT. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY none SP:SAMPLEPREP_PROTOCOL_FILENAME SB_NAD-ATP_Assay_Agilent.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Thermo Hypercarb (50 x 2.1mm, 3um) CH:METHODS_FILENAME SB_NAD-ATP_Assay_Agilent.pdf #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6490 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/mg protein MS_METABOLITE_DATA_START Samples iPSC_01 iPSC_02 iPSC_03 iPSC_04 iPSC_05 iPSC_06 iPSC_07 iPSC_08 iPSC_09 iPSC_10 iPSC_11 iPSC_12 iPSC_13 iPSC_14 iPSC_15 iPSC_16 iPSC_17 Factors Genotype:WT/N370S Genotype:WT/WT Genotype:KO/KO Genotype:WT/N370S Genotype:WT/WT Genotype:WT/N370S Genotype:WT/WT Genotype:KO/KO Genotype:KO/KO Genotype:WT/N370S Genotype:WT/WT Genotype:WT/N370S Genotype:WT/WT Genotype:WT/L444P Genotype:WT/WT Genotype:WT/L444P Genotype:WT/WT 1-Methyl NAM ND 0.64 0.54 NAM 0.73 1.20 0.63 0.69 0.68 0.95 1.29 0.87 0.60 1.08 1.01 2.43 0.96 0.66 NMN ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NAD 2.55 2.74 4.72 1.59 1.90 2.60 2.22 3.51 1.96 0.98 2.21 2.07 3.70 2.32 1.77 1.60 NADP NADH 0.56 0.07 0.28 0.10 0.39 0.22 0.48 0.34 0.23 0.34 0.18 0.25 0.13 0.45 0.22 0.16 NADPH 0.50 0.07 0.12 0.17 0.07 0.37 0.34 0.19 0.36 0.28 0.29 0.26 0.32 0.23 0.54 0.37 0.38 AMP 1.99 5.16 6.24 4.27 3.77 8.33 4.14 13.34 7.78 3.88 11.64 ADP 6.78 18.66 5.35 9.53 5.74 6.38 9.63 9.98 13.53 9.46 4.92 10.57 10.72 22.21 10.42 9.38 5.06 ATP 19.30 32.14 21.20 29.31 7.96 16.49 10.65 10.35 25.73 14.30 2.80 16.22 6.17 42.30 26.10 14.28 GDP 1.62 5.02 1.48 2.33 1.55 1.46 2.29 2.45 2.95 2.21 1.53 2.53 3.04 4.76 2.16 1.99 2.41 GTP 3.97 8.92 4.33 6.20 2.10 3.38 2.67 2.49 4.79 3.19 1.09 3.63 2.07 9.02 5.29 3.15 1.40 CDP 0.53 0.55 0.59 0.55 0.75 1.73 0.78 0.60 CTP 1.79 3.31 2.08 2.76 0.52 1.36 0.83 0.78 1.62 1.21 1.50 4.42 2.66 1.20 UDP 1.26 1.18 1.63 0.92 1.05 1.50 1.74 2.50 1.68 1.22 1.94 2.06 5.25 2.15 1.58 1.08 UTP 6.38 8.60 7.25 9.98 2.22 5.13 2.96 2.98 7.93 4.49 0.91 5.34 2.09 18.78 10.37 4.74 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID NMN 1.13 335.2, 123.1 14180 NAD 1.80 664.2, 136.1 5893 NADP 1.64 744.2, 136.1 5886 NADH 1.86 666.2, 649.2 439153 NADPH 0.66 746.2, 729.2 5884 AMP 1.51 348.1, 136.1 6083 ADP 1.51 428.0, 136.1 6022 ATP 1.50 508.1, 136.1 5957 1-Methyl NAM NAM GDP GTP CDP CTP UDP UTP METABOLITES_END #END