#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