#METABOLOMICS WORKBENCH amitch_20160908_131619 DATATRACK_ID:727 STUDY_ID:ST000468 ANALYSIS_ID:AN000731 PROJECT_ID:PR000360 VERSION 1 CREATED_ON September 12, 2016, 2:48 pm #PROJECT PR:PROJECT_TITLE Enterococcus faecalis nucleotie profiles PR:PROJECT_SUMMARY Determination of metabolite profile of E. faecalis OG1RF control as compared to PR:PROJECT_SUMMARY exposure to mupirocin or decoyinine PR:INSTITUTE Univeristy of Florida PR:DEPARTMENT Oral Biology PR:LABORATORY Oral Biology Lemos-Abranches PR:LAST_NAME Lemos PR:FIRST_NAME Jose PR:ADDRESS 1395 Center Drive, PO Box 100424 Gainesville, FL 32610 PR:EMAIL jlemos@dental.ufl.edu PR:PHONE 352-273-8843 PR:FUNDING_SOURCE UF start-up funds #STUDY ST:STUDY_TITLE Enterococcus faecalis nucleotide profiles following mupirocin or decoyinine ST:STUDY_TYPE Exposure to mupirocin or decoyinine ST:STUDY_SUMMARY Triplicate samples of E. faecalis OG1RF will be grown in FMC-AUG to OD600 = ST:STUDY_SUMMARY 0.25. The cultures will be split and exposed to DMSO, mupirocin (0.01mg/mL) or ST:STUDY_SUMMARY decoyinine (0.1mg/mL) for 15 minutes. 6E9 cells will be harvested by fast ST:STUDY_SUMMARY filtration, scraped off of membrane into ice-cold PBS, and pelleted by ST:STUDY_SUMMARY centrifugation, and stored at -80C until shipment. ST:INSTITUTE Univeristy of Florida ST:DEPARTMENT SECIM ST:LABORATORY Lemos-Abranches ST:LAST_NAME Kajfasz ST:FIRST_NAME Jessica ST:ADDRESS 1395 Center Drive, PO Box 100424 Gainesville, FL 32610 ST:EMAIL jkajfasz@dental.ufl.edu ST:PHONE 352-273-6675 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 9 #SUBJECT SU:SUBJECT_TYPE Bacterial cells SU:SUBJECT_SPECIES Enterococcus faecalis SU:TAXONOMY_ID 1351 SU:GENOTYPE_STRAIN OG1RF #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 1 DMSO OG1RF_1 DMSO Exposure:DMSO SUBJECT_SAMPLE_FACTORS 2 DMSO OG1RF_2 DMSO Exposure:DMSO SUBJECT_SAMPLE_FACTORS 3 DMSO OG1RF_3 DMSO Exposure:DMSO SUBJECT_SAMPLE_FACTORS 1 Mup OG1RF_1 Mup Exposure:Mupirocin, 0.01mg/mL SUBJECT_SAMPLE_FACTORS 2 Mup OG1RF_2 Mup Exposure:Mupirocin, 0.01mg/mL SUBJECT_SAMPLE_FACTORS 3 Mup OG1RF_3 Mup Exposure:Mupirocin, 0.01mg/mL SUBJECT_SAMPLE_FACTORS 1 Decoy OG1RF_1 Decoy Exposure:Decoyinine, 0.1mg/mL SUBJECT_SAMPLE_FACTORS 2 Decoy OG1RF_2 Decoy Exposure:Decoyinine, 0.1mg/mL SUBJECT_SAMPLE_FACTORS 3 Decoy OG1RF_3 Decoy Exposure:Decoyinine, 0.1mg/mL #COLLECTION CO:COLLECTION_SUMMARY After the 15 minute treatment interval, 20mL sample will be harvested by CO:COLLECTION_SUMMARY filtration onto 0.45uM nitrocellulose membrane. The cells will be scraped off of CO:COLLECTION_SUMMARY the membrane into ice-cold PBS (~1mL), centrifuged for 45 seconds at 12K RPM, CO:COLLECTION_SUMMARY liquid aspirated and stored at -80C until sending for analysis. CO:SAMPLE_TYPE Bacterial cells CO:COLLECTION_METHOD Fast filtration CO:VOLUMEORAMOUNT_COLLECTED 20ml CO:STORAGE_CONDITIONS -80C #TREATMENT TR:TREATMENT_SUMMARY Triplicate cultures of E. faecalis OG1RF will be grown in FMC-AUG to OD600 = TR:TREATMENT_SUMMARY 0.25. The cultures will be split into 3 equal volumes of 30mL which will be TR:TREATMENT_SUMMARY exposed to 100uL DMSO, 0.01mg/mL mupirocin, or 0.1mg/mL decoyinine for 15 TR:TREATMENT_SUMMARY minutes. At this point, 10mL sample will be removed for protein analysis. The TR:TREATMENT_SUMMARY remaining 20mL will be harvested by fast filtration for analysis.|Triplicate TR:TREATMENT_SUMMARY cultures of E. faecalis OG1RF will be grown in FMC-AUG to OD600 = 0.25. The TR:TREATMENT_SUMMARY cultures will be split into 3 equal volumes of 30mL which will be exposed to TR:TREATMENT_SUMMARY 100uL DMSO, 0.01mg/mL mupirocin, or 0.1mg/mL decoyinine for 15 minutes. At this TR:TREATMENT_SUMMARY point, 10mL sample will be removed for protein analysis. The remaining 20mL will TR:TREATMENT_SUMMARY be harvested by fast filtration for analysis.|Triplicate cultures of E. faecalis TR:TREATMENT_SUMMARY OG1RF will be grown in FMC-AUG to OD600 = 0.25. The cultures will be split into TR:TREATMENT_SUMMARY 3 equal volumes of 30mL which will be exposed to 100uL DMSO, 0.01mg/mL TR:TREATMENT_SUMMARY mupirocin, or 0.1mg/mL decoyinine for 15 minutes. At this point, 10mL sample TR:TREATMENT_SUMMARY will be removed for protein analysis. The remaining 20mL will be harvested by TR:TREATMENT_SUMMARY fast filtration for analysis. TR:TREATMENT_COMPOUND DMSO|Mupirocin|Decoyinine TR:TREATMENT_DOSE 100uL|0.01mg/mL|0.1mg/mL TR:TREATMENT_DOSEDURATION 15 min|15 min|15 min TR:TREATMENT_VEHICLE DMSO|DMSO|DMSO #SAMPLEPREP SP:SAMPLEPREP_SUMMARY NAD-ATP SP:SAMPLEPREP_PROTOCOL_FILENAME SB_NAD-ATP_Assay_Agilent.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY NAD-ATP 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 AN:ANALYSIS_PROTOCOL_FILE SB_NAD-ATP_Assay_Agilent.pdf #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 OG1RF_1 DMSO OG1RF_2 DMSO OG1RF_3 DMSO OG1RF_1 Mup OG1RF_2 Mup OG1RF_3 Mup OG1RF_1 Decoy OG1RF_2 Decoy OG1RF_3 Decoy Factors Exposure:DMSO Exposure:DMSO Exposure:DMSO Exposure:Mupirocin, 0.01mg/mL Exposure:Mupirocin, 0.01mg/mL Exposure:Mupirocin, 0.01mg/mL Exposure:Decoyinine, 0.1mg/mL Exposure:Decoyinine, 0.1mg/mL Exposure:Decoyinine, 0.1mg/mL NMN 0.05 0.07 0.08 0.04 N.D. 0.07 0.11 0.08 0.13 NAD 2.33 2.72 2.73 3.87 N.D. 4.59 5.54 5.49 6.31 NADP 0.25 0.29 0.31 0.34 N.D. 0.40 0.44 0.42 0.50 AMP 1.63 1.48 1.26 4.33 N.D. 3.87 6.29 3.68 7.80 ADP N.D. 5.58 5.53 ATP 0.58 1.17 1.54 1.50 N.D. 3.07 1.91 4.49 1.93 GDP 0.699144904 0.814446652 0.864344745 0.511195459 N.D. 0.57325359 0.705216686 0.603863247 0.735392147 GTP 0.564649253 0.706891089 N.D. 0.413789023 0.441168195 CDP 0.170140598 0.229450701 0.236300634 0.564743115 N.D. 0.865106823 0.350493204 0.460581436 0.410636444 CTP 0.092262162 0.214914424 0.267655897 0.316088971 N.D. 0.810274935 0.203505352 0.514360372 0.206382691 UDP 1.118681816 1.170668692 0.901959615 1.388781358 N.D. 1.427531646 1.498960983 1.24000226 1.723860369 UTP 0.777514404 1.228959831 1.281240802 1.096361577 N.D. 1.666613932 1.028269484 1.555148577 1.033884632 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID NMN 1.13 335.2 14180 NAD 1.80 664.2 5893 NADP 1.64 744.2 5886 AMP 1.51 348.1 6083 ADP 1.51 428 6022 ATP 1.50 508.1 5957 GDP GTP CDP CTP UDP UTP METABOLITES_END #END