#METABOLOMICS WORKBENCH sakanaka_20190926_011513 DATATRACK_ID:1825 STUDY_ID:ST001260 ANALYSIS_ID:AN002090 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 POSITIVE MS:MS_COMMENTS The conditions for measurement of cationic metabolites were as follows. Run MS:MS_COMMENTS buffer: Cation Buffer Solution (H3301-1001; Human Metabolome Technologies MS:MS_COMMENTS (HMT)), CE voltage: +27kV, MS ionization: ESI positive, MS capillary voltage: MS:MS_COMMENTS 4,000V, MS scan range: m/z 50-1,000, and sheath liquid: HMT Sheath Liquid MS:MS_COMMENTS (H3301-1020). Identification of metabolites and evaluation of the relative MS:MS_COMMENTS amounts were conducted using Master Hands (version 2.16.0.15 and 2.17.1.11; Keio MS:MS_COMMENTS University, Tokyo, Japan) with the HMT metabolite database. The relative amount MS:MS_COMMENTS of each metabolite was calculated with reference to the internal standard MS:MS_COMMENTS 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 N-Acetylornithine 0.000000 0.000000 0.000000 0.001700 0.000971 0.001944 0.000000 0.000000 0.000000 0.000000 0.000287 0.000327 Hypoxanthine 0.000000 0.000000 0.000000 0.000000 0.000000 0.000090 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 Tyramine 0.000000 0.000000 0.000000 0.000000 0.000000 0.000123 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 D-Alanyl-D-alanine 0.000000 0.000000 0.000000 0.000235 0.000204 0.000245 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 N-Acetyllysine 0.000000 0.000000 0.000000 0.000167 0.000195 0.000207 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 Trp 0.000000 0.000000 0.000000 0.000064 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 Ala 0.001197 0.001545 0.001298 0.002398 0.002909 0.004478 0.001419 0.001540 0.002027 0.002711 0.003243 0.003293 Nicotinamide 0.000632 0.000503 0.000367 0.001796 0.001037 0.000700 0.000451 0.000387 0.000222 0.000554 0.000597 0.001113 Putrescine 0.001012 0.004839 0.000000 0.010831 0.007550 0.006383 0.000502 0.000258 0.001038 0.007242 0.002780 0.000688 Cytosine 0.000085 0.000102 0.000087 0.000124 0.000140 0.000204 0.000129 0.000119 0.000118 0.000125 0.000127 0.000113 Guanine 0.000187 0.000323 0.000322 0.000386 0.000439 0.000566 0.000279 0.000306 0.000387 0.000176 0.000272 0.000250 Glu 0.000406 0.000563 0.000565 0.000659 0.000857 0.001044 0.000573 0.000720 0.000999 0.000896 0.001278 0.001529 Adenine 0.001024 0.001218 0.001254 0.001621 0.001946 0.001963 0.001298 0.001255 0.001218 0.001458 0.001608 0.001609 Thymidine 0.005108 0.006825 0.004472 0.006567 0.009453 0.007322 0.005142 0.006168 0.005132 0.005724 0.006030 0.006693 Carnitine 0.000000 0.000086 0.000000 0.000119 0.000124 0.000000 0.000141 0.000000 0.000141 0.000165 0.000000 0.000170 4-Guanidinobutyric acid 0.000000 0.000090 0.000000 0.000000 0.000147 0.000104 0.000000 0.000092 0.000079 0.000000 0.000000 0.000076 Asn 0.000188 0.000000 0.000207 0.000239 0.000000 0.000292 0.000000 0.000000 0.000000 0.000233 0.000265 0.000267 Agmatine 0.000064 0.000605 0.000000 0.000590 0.000449 0.000313 0.000000 0.000000 0.000075 0.000320 0.000127 0.000000 Benzimidazole 0.000000 0.000134 0.000000 0.000105 0.000000 0.000234 0.000117 0.000109 0.000091 0.000000 0.000000 0.000100 Adenosine 0.000966 0.001146 0.001025 0.001252 0.001427 0.001256 0.001271 0.001155 0.001168 0.001204 0.001367 0.001303 Gly 0.000801 0.000888 0.000703 0.001052 0.000785 0.000970 0.000639 0.000900 0.000807 0.000989 0.000841 0.000855 Methionine sulfoxide 0.000442 0.000712 0.000521 0.000411 0.000833 0.000720 0.000527 0.000672 0.000685 0.000541 0.000663 0.000679 Lys 0.001852 0.003023 0.002285 0.002654 0.002705 0.002922 0.003473 0.003247 0.004392 0.004300 0.004119 0.003974 Morpholine 0.000000 0.000239 0.000189 0.000000 0.000222 0.000269 0.000000 0.000000 0.000000 0.000000 0.000195 0.000000 Cadaverine 0.000000 0.000172 0.000000 0.000225 0.000203 0.000156 0.000050 0.000000 0.000195 0.001069 0.000390 0.000097 Deoxyadenosine 0.000254 0.000262 0.000297 0.000296 0.000323 0.000295 0.000353 0.000303 0.000263 0.000301 0.000344 0.000396 Val 0.001477 0.001710 0.001716 0.000000 0.000000 0.001797 0.000000 0.001349 0.001756 0.001520 0.001379 0.001574 Diethanolamine 0.005486 0.003696 0.005038 0.005372 0.004006 0.005957 0.004468 0.004122 0.007336 0.005524 0.004644 0.005985 Citrulline 0.000124 0.000080 0.000105 0.000107 0.000100 0.000119 0.000000 0.000180 0.000110 0.000147 0.000086 0.000093 Tyr 0.000308 0.000453 0.000314 0.000350 0.000361 0.000400 0.000357 0.000462 0.000459 0.000404 0.000379 0.000366 Asp 0.000447 0.000456 0.000421 0.000351 0.000475 0.000516 0.000000 0.000447 0.000502 0.000383 0.000417 0.000413 His 0.000082 0.000107 0.000068 0.000101 0.000073 0.000083 0.000000 0.000083 0.000076 0.000103 0.000000 0.000000 Mevalolactone 0.001394 0.001316 0.001486 0.001423 0.001420 0.001350 0.001786 0.001741 0.001436 0.001525 0.001294 0.001514 Arg 0.000874 0.001456 0.001087 0.001085 0.001103 0.001202 0.001387 0.001220 0.001563 0.000973 0.001051 0.001041 Met 0.001160 0.000959 0.001288 0.001294 0.001070 0.000986 0.001363 0.001075 0.001022 0.001225 0.001256 0.001357 Riboflavin 0.000573 0.000559 0.000864 0.000585 0.000505 0.000860 0.000672 0.000577 0.000742 0.000685 0.001022 0.000748 Pro 0.000366 0.000387 0.000273 0.000337 0.000306 0.000358 0.000281 0.000349 0.000291 0.000319 0.000257 0.000320 Urocanic acid 0.000164 0.000153 0.000142 0.000000 0.000000 0.000149 0.000204 0.000262 0.000160 0.000189 0.000000 0.000147 5-Aminoindole 0.000000 0.000000 0.000117 0.000099 0.000123 0.000000 0.000130 0.000117 0.000109 0.000204 0.000192 0.000152 Ile 0.000787 0.001557 0.001068 0.000952 0.001138 0.001020 0.000952 0.001062 0.001351 0.001097 0.001200 0.001237 Phe 0.000403 0.000721 0.000546 0.000449 0.000519 0.000541 0.000459 0.000642 0.000672 0.000636 0.000569 0.000544 Ethanolamine 0.000973 0.001708 0.002068 0.000940 0.001373 0.001769 0.001912 0.000971 0.002061 0.001222 0.002131 0.001026 Choline 0.000329 0.000394 0.000382 0.000351 0.000317 0.000264 0.000260 0.000258 0.000288 0.000360 0.000252 0.000235 11-Aminoundecanoic acid 0.000697 0.000654 0.000706 0.000611 0.000495 0.000586 0.000944 0.000658 0.000706 0.000670 0.000767 0.000691 Ser 0.001342 0.001206 0.001013 0.001042 0.000891 0.000989 0.000842 0.001248 0.001074 0.001200 0.000835 0.001109 Thr 0.000392 0.000345 0.000335 0.000364 0.000246 0.000244 0.000151 0.000374 0.000294 0.000417 0.000354 0.000318 Urea 0.005337 0.007376 0.004305 0.005505 0.004520 0.003316 0.005156 0.005980 0.004109 0.006400 0.004599 0.005205 Ornithine 0.000406 0.000443 0.000241 0.000307 0.000209 0.000223 0.000180 0.000280 0.000239 0.000365 0.000240 0.000261 Cyclohexylamine 0.000120 0.000152 0.000399 0.000000 0.000000 0.000150 0.000293 0.000127 0.000271 0.000154 0.000270 0.000114 Leu 0.001132 0.004551 0.002356 0.001272 0.001739 0.001853 0.001402 0.001639 0.002178 0.001567 0.001817 0.001862 Triethanolamine 0.000526 0.000748 0.000380 0.000342 0.000290 0.000255 0.000368 0.000539 0.000296 0.000370 0.000237 0.000416 Gln 0.000109 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000174 0.000000 0.000133 0.000000 0.000000 Ribavirin 0.000000 0.000000 0.000590 0.000000 0.000000 0.000000 0.000801 0.000000 0.000000 0.000000 0.000962 0.000000 Uridine 0.000000 0.000000 0.000590 0.000000 0.000000 0.000000 0.000000 0.000000 0.000585 0.000000 0.000961 0.000000 1-Methyl-2-pyrrolidone 0.000000 0.000000 0.002810 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 Thiamine 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000138 0.000095 0.000126 0.000000 0.000057 0.000074 Thymine 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000522 0.000000 0.000000 0.000000 0.000000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID HMDB ID m/z MT N-Acetylornithine C00437 HMDB03357 175.10739 9.4308924 Hypoxanthine C00262 HMDB00157 137.0452 10.786189 Tyramine C00483 HMDB00306 138.09122 8.2844925 D-Alanyl-D-alanine C00993 HMDB03459 161.0907333 9.147895667 N-Acetyllysine C12989 HMDB00446 189.12248 9.634831 Trp C00078 HMDB00929 205.09642 10.764809 Ala C00041 HMDB00161 90.05512925 8.913873375 Nicotinamide C00153 HMDB01406 123.0549668 7.360236567 Putrescine C00134 HMDB01414 89.10758545 4.736842664 Cytosine C00380 HMDB00630 112.0511697 7.230034208 Guanine C00242 HMDB00132 152.0567542 8.235241167 Glu C00025 HMDB00148 148.0603108 10.69181529 Adenine C00147 HMDB00034 136.0614325 7.578604225 Thymidine C00214 HMDB00273 243.0872392 20.29413092 Carnitine C00318 HMDB00062 162.1125486 8.536784143 4-Guanidinobutyric acid C01035 HMDB03464 146.0923617 8.2206355 Asn C00152 HMDB00168 133.0596786 10.25693 Agmatine C00179 HMDB01432 131.12898 5.20133825 Benzimidazole C02009 119.0607514 7.160608286 Adenosine C00212 HMDB00050 268.1034475 9.70177625 Gly C00037 HMDB00123 76.0392545 8.235236333 Methionine sulfoxide C02989 HMDB02005 166.05293 11.52399283 Lys C00047 HMDB00182 147.1122192 6.868533692 Morpholine C14452 HMDB31581 88.075928 6.6193306 Cadaverine C01672 HMDB02322 103.1227122 5.015084356 Deoxyadenosine C00559 HMDB00101 252.10852 9.5298475 Val C00183 HMDB00883 118.0860323 9.8469045 Diethanolamine C06772 HMDB04437 106.0862033 7.606009075 Citrulline C00327 HMDB00904 176.1025764 10.79440164 Tyr C00082 HMDB00158 182.080525 11.06779121 Asp C00049 HMDB00191 134.0441427 11.22503473 His C00135 HMDB00177 156.0760644 7.262436278 Mevalolactone HMDB06024 131.07069 20.38325633 Arg C00062 HMDB00517 175.118755 7.091283883 Met C00073 HMDB00696 150.0582575 10.49095625 Riboflavin C00255 HMDB00244 377.1439858 20.18657542 Pro C00148 HMDB00162 116.0708242 10.56531775 Urocanic acid C00785 HMDB00301 139.0500878 8.166587778 5-Aminoindole 133.0757433 7.730951111 Ile C00407 HMDB00172 132.1018067 10.02844742 Phe C00079 HMDB00159 166.08573 10.83110988 Ethanolamine C00189 HMDB00149 62.06036983 6.3475439 Choline C00114 HMDB00097 104.1067767 6.852281442 11-Aminoundecanoic acid C19325 202.1797783 9.532430625 Ser C00065 HMDB00187 106.0497429 9.799276083 Thr C00188 HMDB00167 120.0654325 10.28579879 Urea C00086 HMDB00294 61.04034792 19.48782192 Ornithine C00077 HMDB00214 133.0970833 6.802580233 Cyclohexylamine C00571 100.1118706 7.659856 Leu C00123 HMDB00687 132.1019008 10.12249429 Triethanolamine C06771 150.1118542 8.178730667 Gln C00064 HMDB00641 147.07592 10.515503 Ribavirin C07234 245.0900233 20.27269667 Uridine C00299 HMDB00296 245.07693 20.145361 1-Methyl-2-pyrrolidone C11118 100.07619 20.041794 Thiamine C00378 HMDB00235 265.1111 6.60267816 Thymine C00178 HMDB00262 127.04963 20.304962 METABOLITES_END #END