#METABOLOMICS WORKBENCH sakanaka_20190926_011513 DATATRACK_ID:1825 STUDY_ID:ST001260 ANALYSIS_ID:AN002091 PROJECT_ID:PR000846 VERSION 1 CREATED_ON October 2, 2019, 9:13 am #PROJECT PR:PROJECT_TITLE Fusobacterium nucleatum metabolome PR:PROJECT_SUMMARY CE-TOFMS-based untargeted analysis of the intracellular 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 Fusobacterium nucleatum when co-cultured with other oral ST:STUDY_TITLE microbes (part-I) ST:STUDY_SUMMARY We used membrane-separated co-culture systems to globally assess metabolomic ST:STUDY_SUMMARY changes of Fusobacterium nucleatum when co-cultured with Streptococcus gordonii ST:STUDY_SUMMARY 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 Bacteria SU:SUBJECT_SPECIES Fusobacterium nucleatum subsp. nucleatum ATCC 25586 SU:TAXONOMY_ID 190304 #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 After 6 h of co-culture, F. nucleatum cells were collected by pipetting from the CO:COLLECTION_SUMMARY lower chamber and washed with Milli-Q water by centrifugation. Bacterial pellets CO:COLLECTION_SUMMARY were immediately fixed by adding methanol containing 5 µM internal standard. 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 (H3302-1023), 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 Isovaleric acid 0.000000 0.000000 0.000000 0.001358 0.000000 0.001127 0.001376 0.001464 0.000000 0.001204 0.000000 0.000000 Heptanoic acid 0.000000 0.000000 0.000000 0.000632 0.000596 0.000000 0.000000 0.000000 0.000000 0.000379 0.000000 0.000000 Sedoheptulose 7-phosphate 0.000000 0.000000 0.000000 0.000000 0.000000 0.000377 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 dTMP 0.000000 0.000000 0.000000 0.000000 0.000000 0.000262 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 UMP 0.000000 0.000000 0.000000 0.000000 0.000292 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 AMP 0.000965 0.001390 0.000921 0.003579 0.003365 0.002824 0.000608 0.000658 0.000619 0.001669 0.001632 0.001693 NAD+ 0.000571 0.000443 0.000728 0.001089 0.001201 0.001658 0.000694 0.000679 0.000791 0.000742 0.001158 0.000828 Decanoic acid 0.001530 0.001790 0.001149 0.002379 0.002445 0.001720 0.002092 0.001779 0.001170 0.002078 0.000882 0.002270 Hexanoic acid 0.000912 0.001072 0.000834 0.001746 0.001352 0.000970 0.001134 0.001225 0.000919 0.001120 0.000997 0.000881 2-Hydroxyvaleric acid 0.000416 0.000638 0.000679 0.000997 0.000825 0.000589 0.001023 0.000867 0.000734 0.000815 0.000000 0.000623 Octanoic acid 0.000977 0.001298 0.000855 0.001678 0.001551 0.001022 0.000959 0.001042 0.000621 0.001132 0.000000 0.001032 Glucose 6-phosphate 0.000437 0.000619 0.000722 0.000733 0.000707 0.000966 0.000608 0.000753 0.000605 0.000586 0.000493 0.000507 Pelargonic acid 0.000562 0.000000 0.000000 0.000758 0.000000 0.000000 0.000000 0.000883 0.000000 0.000000 0.000000 0.000000 Quinic acid 0.000541 0.000395 0.000000 0.000892 0.000370 0.000000 0.000000 0.000000 0.000000 0.000714 0.000000 0.000557 Flavin adenine dinucleotide 0.001566 0.001180 0.002385 0.001672 0.002329 0.002695 0.001931 0.002125 0.001855 0.001835 0.002835 0.002168 Lauric acid 0.019638 0.029437 0.020191 0.025143 0.027660 0.031016 0.028379 0.026942 0.021742 0.022735 0.021155 0.028440 3-Phenylpropionic acid 0.000375 0.000679 0.000597 0.000628 0.000725 0.000563 0.000724 0.000727 0.000489 0.000770 0.000432 0.000675 ADP 0.001195 0.000794 0.001659 0.000953 0.001698 0.001497 0.001571 0.001769 0.001545 0.001081 0.001404 0.002108 Terephthalic acid 0.001144 0.000876 0.000850 0.001202 0.000927 0.001124 0.001322 0.001159 0.000922 0.000851 0.000910 0.001151 Flavin mononucleotide 0.003893 0.003594 0.004566 0.004733 0.003969 0.004557 0.004148 0.003115 0.003713 0.004700 0.005776 0.004572 Lactic acid 0.007329 0.011481 0.006691 0.010364 0.009224 0.006099 0.008312 0.007431 0.005224 0.010405 0.005350 0.007540 Dihydroxyacetone phosphate 0.000000 0.000000 0.000741 0.000671 0.000558 0.000849 0.001047 0.000784 0.001063 0.000000 0.000884 0.000000 Myristoleic acid 0.000724 0.000587 0.001115 0.000689 0.000454 0.000794 0.001419 0.001383 0.001059 0.000549 0.000983 0.001257 Glycerol 3-phosphate 0.001583 0.001629 0.001218 0.001160 0.000999 0.000998 0.001242 0.001245 0.000976 0.000929 0.001056 0.000868 Benzoic acid 0.001105 0.001285 0.000000 0.000000 0.000000 0.000000 0.001283 0.001696 0.000000 0.000000 0.000000 0.000000 5-Oxoproline 0.000441 0.000572 0.000000 0.000000 0.000000 0.000000 0.000000 0.000292 0.000000 0.000624 0.000000 0.000000 Glyceraldehyde 3-phosphate 0.000000 0.000000 0.000349 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000320 5-Oxohexanoic acid 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000999 0.000764 0.000000 0.000000 0.000000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID HMDB ID m/z MT Isovaleric acid C08262 HMDB00718 101.0616388 8.8371834 Heptanoic acid HMDB00666 129.09239 8.206988333 Sedoheptulose 7-phosphate C05382 HMDB01068 289.03384 9.194174 dTMP C00364 HMDB01227 321.05112 9.160733 UMP C00105 HMDB00288 323.0299 9.390243 AMP C00020 HMDB00045 346.0568242 8.846265583 NAD+ C00003 HMDB00902 662.1030592 6.235059758 Decanoic acid C01571 HMDB00511 171.1394908 7.574135883 Hexanoic acid C01585 HMDB00535 115.0769255 8.522434667 2-Hydroxyvaleric acid HMDB01863 117.0559795 8.724931045 Octanoic acid C06423 HMDB00482 143.1081927 7.957609736 Glucose 6-phosphate C00668 HMDB01401 259.0239142 9.42149875 Pelargonic acid C01601 HMDB00847 157.12334 7.684384267 Quinic acid C00296 HMDB03072 191.0569883 7.715836183 Flavin adenine dinucleotide C00016 HMDB01248 391.5727842 7.486915667 Lauric acid C02679 HMDB00638 199.1716717 7.291103458 3-Phenylpropionic acid C05629 HMDB00764 149.0604833 8.3869645 ADP C00008 HMDB01341 426.0236725 10.41607963 Terephthalic acid C06337 HMDB02428 165.0197483 16.35139942 Flavin mononucleotide C00061 HMDB01520 455.0992458 7.952716567 Lactic acid C00186 HMDB00190 89.02549667 10.32700825 Dihydroxyacetone phosphate C00111 HMDB01473 168.9920325 12.24683125 Myristoleic acid C08322 HMDB02000 225.1869658 7.1148007 Glycerol 3-phosphate C00093 HMDB00126 171.0068908 11.70746775 Benzoic acid C00180 HMDB01870 121.0302688 9.43087725 5-Oxoproline C01879 HMDB00267 128.035305 9.0731555 Glyceraldehyde 3-phosphate C00118 HMDB01112 168.99168 11.2315135 5-Oxohexanoic acid C02129 129.05674 8.685487 METABOLITES_END #END