#METABOLOMICS WORKBENCH sakanaka_20190926_042826_mwtab.txt DATATRACK_ID:1826 STUDY_ID:ST001261 ANALYSIS_ID:AN002093 PROJECT_ID:PR000847 VERSION 1 CREATED_ON October 2, 2019, 9:23 am #PROJECT PR:PROJECT_TITLE Fusobacterium nucleatum metabolome PR:PROJECT_SUMMARY CE-TOFMS-based untargeted analysis of the extracellular metabolite changes of F. PR:PROJECT_SUMMARY nucleatum when co-cultured with other oral microbes PR:INSTITUTE Osaka University Graduate School of Dentistry PR:DEPARTMENT Department of Preventive Dentistry PR:LAST_NAME Kuboniwa PR:FIRST_NAME Masae PR:ADDRESS Yamadaoka 1-8 PR:EMAIL kuboniwa@dent.osaka-u.ac.jp PR:PHONE 81668792922 #STUDY ST:STUDY_TITLE Metabolic changes of culture supernatants of Fusobacterium nucleatum co-cultured ST:STUDY_TITLE with other oral microbes (part-II) ST:STUDY_SUMMARY We used membrane-separated co-culture systems to globally assess extracellular ST:STUDY_SUMMARY metabolomic changes of Fusobacterium nucleatum co-cultured with Streptococcus ST:STUDY_SUMMARY gordonii and/or Veillonella parvula. ST:INSTITUTE Osaka University Graduate School of Dentistry ST:DEPARTMENT Department of Preventive Dentistry ST:LAST_NAME Kuboniwa ST:FIRST_NAME Masae ST:ADDRESS Yamadaoka 1-8 ST:EMAIL kuboniwa@dent.osaka-u.ac.jp ST:PHONE 81668792922 #SUBJECT SU:SUBJECT_TYPE Other SU:SUBJECT_SPECIES Fusobacterium nucleatum #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Fn_1 partner:none SUBJECT_SAMPLE_FACTORS - Fn_2 partner:none SUBJECT_SAMPLE_FACTORS - Fn_3 partner:none SUBJECT_SAMPLE_FACTORS - Fn-Sg_1 partner:Sg SUBJECT_SAMPLE_FACTORS - Fn-Sg_2 partner:Sg SUBJECT_SAMPLE_FACTORS - Fn-Sg_3 partner:Sg SUBJECT_SAMPLE_FACTORS - Fn-Vp_1 partner:Vp SUBJECT_SAMPLE_FACTORS - Fn-Vp_2 partner:Vp SUBJECT_SAMPLE_FACTORS - Fn-Vp_3 partner:Vp SUBJECT_SAMPLE_FACTORS - Fn-SgVp_1 partner:SgVp SUBJECT_SAMPLE_FACTORS - Fn-SgVp_2 partner:SgVp SUBJECT_SAMPLE_FACTORS - Fn-SgVp_3 partner:SgVp #COLLECTION CO:COLLECTION_SUMMARY Spent medium from cultures and sterile CDM were centrifuged, filtered through CO:COLLECTION_SUMMARY 0.22-µm filtration devices (Millipore) and lyophilized. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY Co-culture growth was performed by inoculating 1.4E+10 cells of F. nucleatum in TR:TREATMENT_SUMMARY CDM in the lower chamber of a Transwell unit with 0.4-µm pore polystyrene TR:TREATMENT_SUMMARY membrane inserts (Corning, NY, USA), into which 1.4E+10 cells of S. gordonii, V. TR:TREATMENT_SUMMARY parvula or their mixture (7E+9 cells each) in CDM, or an equal volume of CDM (as TR:TREATMENT_SUMMARY a control) were added. The setup was anaerobically incubated in triplicate for TR:TREATMENT_SUMMARY 37°C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To remove protein, 2 ml of chloroform and 0.8 ml of ultrapure water were added SP:SAMPLEPREP_SUMMARY to the samples, which were thoroughly mixed and centrifuged at 2300 × g for 5 SP:SAMPLEPREP_SUMMARY minutes at 4˚C. The upper aqueous layer was then transferred to ultrafilter SP:SAMPLEPREP_SUMMARY tips (Amicon ultrafilter system™) and centrifuged at 9100 × g for 120 minutes SP:SAMPLEPREP_SUMMARY at 4˚C. Filtered material was dried under reduced pressure, followed by SP:SAMPLEPREP_SUMMARY suspension in 50 µl of ultrapure water. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE CE CH:INSTRUMENT_NAME Agilent 6210 CH:COLUMN_NAME None #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6210 TOF MS:INSTRUMENT_TYPE CE-TOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The conditions for measurement of anionic metabolites were as follows. Run MS:MS_COMMENTS buffer: Anion Buffer Solution (I3302-1023), CE voltage: +30kV, MS ionization: MS:MS_COMMENTS ESI negative, MS capillary voltage: 3,500V, MS scan range: m/z 50-1,000, and MS:MS_COMMENTS sheath liquid: HMT Sheath Liquid (H3301-1020). Identification of metabolites and MS:MS_COMMENTS evaluation of the relative amounts were conducted using Master Hands (version MS:MS_COMMENTS 2.16.0.15 and 2.17.1.11; Keio University, Tokyo, Japan) with the HMT metabolite MS:MS_COMMENTS database. The relative amount of each metabolite was calculated with reference MS:MS_COMMENTS to the internal standard material (HMT). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples Fn_1 Fn_2 Fn_3 Fn-Sg_1 Fn-Sg_2 Fn-Sg_3 Fn-Vp_1 Fn-Vp_2 Fn-Vp_3 Fn-SgVp_1 Fn-SgVp_2 Fn-SgVp_3 Factors partner:none partner:none partner:none partner:Sg partner:Sg partner:Sg partner:Vp partner:Vp partner:Vp partner:SgVp partner:SgVp partner:SgVp 5-Oxoproline 0.0000000 0.0000000 0.0000000 0.0000718 0.0000941 0.0000781 0.0000000 0.0000000 0.0000495 0.0000869 0.0000983 0.0001049 2-Hydroxy-4-methylpentanoic acid 0.0000000 0.0000000 0.0000000 0.0000444 0.0000525 0.0000000 0.0000742 0.0000867 0.0000818 0.0001794 0.0001587 0.0001838 Fructose 1,6-diphosphate 0.0000000 0.0000000 0.0000000 0.0000319 0.0000000 0.0000000 0.0000000 0.0000000 0.0000325 0.0000000 0.0000000 0.0000000 Lactic acid 0.0000000 0.0000000 0.0000000 0.0291934 0.0302548 0.0327572 0.0000000 0.0000000 0.0000000 0.0003831 0.0004416 0.0004751 2-Hydroxybutyric acid 0.0000000 0.0000000 0.0000000 0.0003324 0.0003332 0.0003354 0.0000000 0.0000000 0.0000000 0.0002448 0.0002334 0.0002089 Glyceric acid 0.0000000 0.0000000 0.0000000 0.0000698 0.0000881 0.0001009 0.0000000 0.0000000 0.0000000 0.0000913 0.0000715 0.0000850 Threonic acid 0.0000000 0.0000000 0.0000000 0.0000320 0.0000377 0.0000000 0.0000000 0.0000000 0.0000000 0.0000366 0.0000467 0.0000530 3-Phosphoglyceric acid 0.0000000 0.0000000 0.0000000 0.0001546 0.0001619 0.0001889 0.0000000 0.0000000 0.0000000 0.0001995 0.0001827 0.0001828 ADP 0.0000000 0.0000000 0.0000000 0.0000308 0.0000382 0.0000380 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 Butyric acid 0.0000307 0.0000284 0.0000307 0.0002834 0.0002581 0.0003055 0.0000769 0.0000820 0.0000882 0.0001541 0.0001416 0.0002825 Ribulose 5-phosphate 0.0002211 0.0002347 0.0002063 0.0005454 0.0005824 0.0005401 0.0003657 0.0003729 0.0003965 0.0007337 0.0006631 0.0006786 Glycerol 3-phosphate 0.0002844 0.0003435 0.0002776 0.0006191 0.0007406 0.0006834 0.0003822 0.0004061 0.0004087 0.0006759 0.0006628 0.0006500 Fructose 6-phosphate 0.0001509 0.0001479 0.0001740 0.0001893 0.0002042 0.0001729 0.0001946 0.0002037 0.0001665 0.0002178 0.0001555 0.0002131 2-Hydroxyvaleric acid 0.0006227 0.0006492 0.0006143 0.0006858 0.0007013 0.0006683 0.0006310 0.0005724 0.0005821 0.0005751 0.0005767 0.0005500 Glucose 6-phosphate 0.0005120 0.0005938 0.0005166 0.0005786 0.0005654 0.0005963 0.0005727 0.0005790 0.0005688 0.0006755 0.0007449 0.0006998 dTMP 0.0000460 0.0000401 0.0000000 0.0000000 0.0000465 0.0000414 0.0000545 0.0000596 0.0000434 0.0000466 0.0000502 0.0000513 dAMP 0.0000361 0.0000369 0.0000384 0.0000431 0.0000000 0.0000313 0.0000584 0.0000509 0.0000446 0.0000000 0.0000418 0.0000441 Dihydroxyacetone phosphate 0.0003718 0.0003715 0.0003333 0.0003110 0.0003546 0.0003596 0.0003396 0.0003481 0.0003549 0.0002839 0.0002951 0.0003243 Sedoheptulose 7-phosphate 0.0012729 0.0013955 0.0013492 0.0010249 0.0011058 0.0010224 0.0013324 0.0015183 0.0013593 0.0010752 0.0011269 0.0011132 Glucose 1-phosphate 0.0002582 0.0002554 0.0002453 0.0001757 0.0001946 0.0002020 0.0002612 0.0002614 0.0002184 0.0001864 0.0001772 0.0001846 NAD+ 0.0000655 0.0000826 0.0000774 0.0000483 0.0000475 0.0000664 0.0000739 0.0000633 0.0000807 0.0000390 0.0000578 0.0000608 UMP 0.0001919 0.0002023 0.0001877 0.0001056 0.0000893 0.0001065 0.0002192 0.0002366 0.0002051 0.0000857 0.0000951 0.0001113 CMP 0.0002997 0.0003451 0.0003100 0.0001484 0.0001556 0.0001603 0.0003341 0.0003791 0.0003479 0.0001258 0.0001298 0.0001526 AMP 0.0012053 0.0013357 0.0012383 0.0006055 0.0005914 0.0005844 0.0013019 0.0014625 0.0013082 0.0005099 0.0004968 0.0005618 GMP 0.0001517 0.0001697 0.0001474 0.0000759 0.0000766 0.0000673 0.0001588 0.0001786 0.0001480 0.0000752 0.0000801 0.0000904 Ribose 5-phosphate 0.0015964 0.0017068 0.0016242 0.0006798 0.0005875 0.0006175 0.0015553 0.0015195 0.0013672 0.0007074 0.0007625 0.0007624 UDP 0.0000932 0.0000956 0.0000857 0.0000330 0.0000000 0.0000000 0.0001171 0.0001033 0.0001033 0.0000547 0.0000000 0.0000495 GDP 0.0001241 0.0001298 0.0001128 0.0000000 0.0000411 0.0000000 0.0001515 0.0001528 0.0001377 0.0000572 0.0000521 0.0000645 CDP 0.0000501 0.0000595 0.0000505 0.0000000 0.0000000 0.0000000 0.0000579 0.0000787 0.0000687 0.0000000 0.0000320 0.0000000 3-Hydroxybutyric acid 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000537 0.0000000 0.0000458 6-Phosphogluconic acid 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000329 0.0000000 0.0000000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID HMDB ID m/z MT 5-Oxoproline C01879 HMDB00267 128.0354243 9.361879357 2-Hydroxy-4-methylpentanoic acid C03264 HMDB00624 131.072555 8.739231 Fructose 1,6-diphosphate C00354 HMDB01058 338.9901 14.5332775 Lactic acid C00186 HMDB00190 89.02537917 10.67127117 2-Hydroxybutyric acid C05984 HMDB00008 103.0404392 9.650622917 Glyceric acid C00258 HMDB00139 105.0198533 10.26958683 Threonic acid C01620 HMDB00943 135.030722 9.1071946 3-Phosphoglyceric acid C00197 HMDB00807 184.9861067 19.97325383 ADP C00008 HMDB01341 426.0245 10.74520867 Butyric acid C00246 HMDB00039 87.0456089 9.73730275 Ribulose 5-phosphate C00199 HMDB00618 229.012835 10.86026113 Glycerol 3-phosphate C00093 HMDB00126 171.0072567 12.04548033 Fructose 6-phosphate C05345 HMDB00124 259.0238158 9.831881417 2-Hydroxyvaleric acid HMDB01863 117.0564091 9.0694405 Glucose 6-phosphate C00668 HMDB01401 259.0238075 9.717618167 dTMP C00364 HMDB01227 321.049514 9.4138964 dAMP C00380 HMDB00630 112.0504596 7.446855308 Dihydroxyacetone phosphate C00111 HMDB01473 168.9914608 12.6090115 Sedoheptulose 7-phosphate C05382 HMDB01068 289.034025 9.483557917 Glucose 1-phosphate C00103 HMDB01586 259.0243158 9.995262583 NAD+ C00003 HMDB00902 662.1039633 6.419506917 UMP C00105 HMDB00288 323.0303858 9.649837667 CMP C00055 HMDB00095 322.0467442 9.45386975 AMP C00020 HMDB00045 346.0569042 9.124236875 GMP C00144 HMDB01397 362.0525633 8.996154667 Ribose 5-phosphate C00117 HMDB01548 229.0128483 10.45055533 UDP C00015 HMDB00295 402.9962589 11.55836039 GDP C00035 HMDB01201 442.018044 10.509797 CDP C00112 HMDB01546 402.0113829 11.3612665 3-Hydroxybutyric acid C01089 HMDB00011 103.0404875 9.3363285 6-Phosphogluconic acid C03194 HMDB12136 76.075815 7.21419765 METABOLITES_END #END