#METABOLOMICS WORKBENCH amitch_20160908_112213 DATATRACK_ID:726 STUDY_ID:ST000466 ANALYSIS_ID:AN000728 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 Amino acids CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6490 CH:COLUMN_NAME Waters AccQ Tag (100 x 2mm, 1.7um) CH:METHODS_FILENAME SB_AA_Assay.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 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 Alanine 13.4968173 13.77484535 17.08474808 11.44786287 29.57921822 27.80150967 Arginine 0.727122952 0.93160068 1.487828796 1.178842015 13.91608989 16.93600365 Asparagine 1.279478236 1.628887762 2.069656943 2.224827051 3.26275759 7.785364418 Aspartic Acid 4.455220825 6.984646068 9.881306129 5.568899683 20.26276077 19.34355353 Citrulline 6.310064266 8.581588219 8.964833713 6.238985801 0.176973847 0.131098646 Glutamic Acid 17.10642159 24.37329968 21.11184161 15.2096646 92.45672307 49.25100429 Glutamine 2.531073596 7.180627198 5.92838685 3.511570294 6.258616833 32.17249124 Glycine 4.404880679 3.490639013 6.156741627 3.869487452 7.282333173 7.485359965 Histidine 0.586065066 0.637094911 0.833566217 0.640196052 1.007666776 1.454040575 Isoleucine 1.392103942 1.917462191 2.249132637 1.519424174 2.00070069 3.156461297 Leucine 4.119419121 7.778756768 6.827787797 4.803071231 10.34482415 12.07748425 Lysine 8.086046824 12.85291899 16.39849601 8.784116838 24.36911396 28.16432389 Methionine 0.964918644 1.249392478 1.421942208 0.976152554 1.442880753 2.198434199 Ornithine 3.072268435 3.612224813 4.246898133 2.245134481 0.368238481 0.451957404 Phenylalanine 1.52883581 1.889360051 2.492716603 1.422059683 2.041916121 2.798384215 Proline 1.450537447 1.050247783 1.117216511 0.671405546 2.036416928 1.908432913 Serine 1.201237763 2.257184921 2.551346905 1.775937567 2.378835817 3.716613361 Threonine 2.707296961 3.475857255 4.598548885 2.550898527 8.081660248 7.673625587 Tryptophan 0.378391222 0.390840699 0.464721524 0.353727692 0.471037662 0.60111904 Tyrosine 0.644789168 0.693982476 0.965012788 0.615679833 0.961151331 0.966145347 Valine 2.021256555 3.032133437 3.323429924 1.812314893 1.869078353 1.427014083 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID Alanine 4.16 260.17 5950 Arginine 2.68 345.21 6322 Asparagine 2.25 303.13 6267 Aspartic Acid 2.99 304.11 5960 Citrulline 3.33 346.21 9750 Glutamic Acid 3.35 318.11 33032 Glutamine 2.65 317.21 5961 Glycine 2.81 246.08 750 Histidine 2.07 326.21 6274 Isoleucine 8.15 302.21 6306 Leucine 8.26 302.21 6106 Lysine 6.66 487.21 5962 Methionine 6.87 320.21 6137 Ornithine 5.93 473.22 6262 Phenylalanine 8.40 336.21 6140 Proline 4.84 286.16 145742 Serine 2.60 276.11 5951 Threonine 3.74 290.11 6288 Tryptophan 8.54 375.21 6305 Tyrosine 6.66 352.21 6057 Valine 7.09 288.21 6287 METABOLITES_END #END