#METABOLOMICS WORKBENCH amitch_20160908_112213 DATATRACK_ID:726 STUDY_ID:ST000466 ANALYSIS_ID:AN000729 PROJECT_ID:PR000358 VERSION 1 CREATED_ON September 12, 2016, 1:31 pm #PROJECT PR:PROJECT_TITLE Streptococcus mutans Amino Acid/Organic Acid Profile PR:PROJECT_SUMMARY Determination of metabolite profiles of S. mutans strains UA159 (WT) and ΔspxA1 PR:PROJECT_SUMMARY in response to oxidative stress PR:INSTITUTE Univeristy of Florida PR:DEPARTMENT Oral Biology PR:LABORATORY 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 Streptococcus mutans organic acids/amino acid profiles, effect of oxidative ST:STUDY_TITLE stress; pilot ST:STUDY_TYPE Amino acid/organic acid profiles of wild-type and mutant strain in response to ST:STUDY_TYPE H2O2 treatment over time. ST:STUDY_SUMMARY S. mutans UA159 and ΔspxA1 will be grown in BHI to OD600 = 0.4. Each culture ST:STUDY_SUMMARY will be split in to 4 samples. For the first sample, 3E10 cells will immediately ST:STUDY_SUMMARY be harvested by fast filtration, scraped off of membrane into ice-cold PBS, and ST:STUDY_SUMMARY pelleted by centrifugation. The remaining three samples will be treated with ST:STUDY_SUMMARY H2O2 to 0.5 mM, and the harvesting process repeated after 15, 30 and 60 minutes. ST:INSTITUTE University of Florida ST:DEPARTMENT SECIM ST:LABORATORY Lemos-Abranches ST:LAST_NAME Hardin ST:FIRST_NAME Emily ST:ADDRESS 1395 Center Drive, PO Box 100424, Gainesville, FL 32610 ST:EMAIL ehardin@ufl.edu ST:PHONE 352-273-8844 ST:NUM_GROUPS 6 ST:TOTAL_SUBJECTS 6 #SUBJECT SU:SUBJECT_TYPE Bacterial cells SU:SUBJECT_SPECIES Streptococcus mutans SU:TAXONOMY_ID 1309 SU:GENOTYPE_STRAIN UA159 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS UA159_0 Lemos_010 Strain:WT | Exposure to H2O2:0.00 SUBJECT_SAMPLE_FACTORS UA159_15min Lemos_011 Strain:WT | Exposure to H2O2:15 min SUBJECT_SAMPLE_FACTORS UA159_30 min Lemos_012 Strain:WT | Exposure to H2O2:30 min SUBJECT_SAMPLE_FACTORS UA159_60 min Lemos_013 Strain:WT | Exposure to H2O2:60 min SUBJECT_SAMPLE_FACTORS spxA1_0 Lemos_014 Strain:ΔspxA1 | Exposure to H2O2:0.00 SUBJECT_SAMPLE_FACTORS spxA1_60 min Lemos_017 Strain:ΔspxA1 | Exposure to H2O2:60 min #COLLECTION CO:COLLECTION_SUMMARY Upon reaching OD600 = 0.4, the culture is split into 4 samples. Three are CO:COLLECTION_SUMMARY treated with H2O2 (0.5 mM). The other is harvested by filtration onto 0.45uM CO:COLLECTION_SUMMARY nitrocellulose membrane. The cells will be scraped off of the membrane into CO:COLLECTION_SUMMARY ice-cold PBS (~1 mL), centrifuged for 30 seconds at 12K RPM, liquid aspirated CO:COLLECTION_SUMMARY and stored at -80C until sending for analysis. This process will be repeated CO:COLLECTION_SUMMARY with the remaining three samples after 15, 30 and 60 minutes treatment with CO:COLLECTION_SUMMARY H2O2. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY 0.5 mM H2O2 for 15, 30 and 60 minutes. Control has 0 H2O2 added. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Amino acids|Organic acids SP:SAMPLEPREP_PROTOCOL_FILENAME SB_AA_Assay.pdf;SB_OA_Assay.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Organic acids CH:CHROMATOGRAPHY_TYPE Reversed phase MS:INSTRUMENT_NAME Thermo Quantiva QQQ CH:COLUMN_NAME Waters Acquity BEH C18 (100 x 2mm, 1.7um) CH:METHODS_FILENAME SB_OA_Assay.pdf #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Quantiva 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 Lemos_010 Lemos_011 Lemos_012 Lemos_013 Lemos_014 Lemos_017 Factors Strain:WT | Exposure to H2O2:0.00 Strain:WT | Exposure to H2O2:15 min Strain:WT | Exposure to H2O2:30 min Strain:WT | Exposure to H2O2:60 min Strain:ΔspxA1 | Exposure to H2O2:0.00 Strain:ΔspxA1 | Exposure to H2O2:60 min Lactate 38.11410827 37.92547892 34.49595534 12.75296215 56.07148179 53.19175841 Pyruvate 1.518795621 1.768367821 1.500451299 0.745049277 2.55928227 2.568756137 Succinate 0.46892125 0.476350026 0.554115095 0.44869083 0.531215577 0.224579712 Fumarate 0.010669337 0.06533411 0.103272586 0.078208839 0.117524568 0.104726182 Malate a-KG 0.085819743 0.075291412 0.087048149 0.062269822 1.175718779 0.34426083 Citrate 0.409795849 0.607818473 0.671669804 0.396595419 0.323044238 0.369449066 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID Lactate 3.98 196.1 107689 Pyruvate 6.68 299.1 1060 Succinate 5.44 329.1 1110 Fumarate 5.59 327.1 444972 Malate 5.28 345.1 525 a-KG 6.59 462.2 164533 Citrate 5.95 508.25 311 METABOLITES_END #END