#METABOLOMICS WORKBENCH Javier_Munoz_Briones_24_20230930_161810 DATATRACK_ID:4352 STUDY_ID:ST002908 ANALYSIS_ID:AN004772 PROJECT_ID:PR001808
VERSION             	1
CREATED_ON             	October 3, 2023, 9:44 am
#PROJECT
PR:PROJECT_TITLE                 	Rumenomics: Evaluation of rumen metabolites content on phenotypical features of
PR:PROJECT_TITLE                 	sheep
PR:PROJECT_TYPE                  	Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy
PR:PROJECT_TYPE                  	(UPLC-MS/MS)
PR:PROJECT_SUMMARY               	Study the link between the gut microbiome and glycosaminoglycan (GAG) production
PR:PROJECT_SUMMARY               	and turnover in synovial joint tissues using cell culture, tissue biomechanics,
PR:PROJECT_SUMMARY               	and studies in mice and sheep. Gut-Joint Axis. Here we evaluate the functional
PR:PROJECT_SUMMARY               	linkages between 510 metabolites collected from rumen compartment in sheep and
PR:PROJECT_SUMMARY               	identified from Metabolomics with phenotypic features such as weight, sex, age
PR:PROJECT_SUMMARY               	and breed. The associations between metabolites and phenotypic features were
PR:PROJECT_SUMMARY               	established by principal component analysis, partial least squares regression,
PR:PROJECT_SUMMARY               	univariate linear modeling and multiple hypothesis testing. With these results,
PR:PROJECT_SUMMARY               	we extract association insights that reveal the most influential metabolites per
PR:PROJECT_SUMMARY               	each phenotypic feature.
PR:INSTITUTE                     	Purdue University
PR:DEPARTMENT                    	Biomedical Engineering - Veterinary Clinical Sciences
PR:LAST_NAME                     	Munoz-Briones
PR:FIRST_NAME                    	Javier
PR:ADDRESS                       	610 Purdue Mall, West Lafayette, IN 47907
PR:EMAIL                         	munoz99@purdue.edu
PR:PHONE                         	7657469102
PR:FUNDING_SOURCE                	DARPA
PR:CONTRIBUTORS                  	Smrutiti Jena, Brendan Ball, Tim Lescun, Deva Chan, Doug Brubaker, Javier Munoz
PR:CONTRIBUTORS                  	Briones
#STUDY
ST:STUDY_TITLE                   	Rumenomics: Evaluation of rumen metabolites content on phenotypical features of
ST:STUDY_TITLE                   	sheep
ST:STUDY_SUMMARY                 	Study the link between the gut microbiome and glycosaminoglycan (GAG) production
ST:STUDY_SUMMARY                 	and turnover in synovial joint tissues using cell culture, tissue biomechanics,
ST:STUDY_SUMMARY                 	and studies in mice and sheep. Gut-Joint Axis. Here we evaluate the functional
ST:STUDY_SUMMARY                 	linkages between 510 metabolites collected from rumen compartment in sheep and
ST:STUDY_SUMMARY                 	identified from Metabolomics with phenotypic features such as weight, sex, age
ST:STUDY_SUMMARY                 	and breed. The associations between metabolites and phenotypic features were
ST:STUDY_SUMMARY                 	established by principal component analysis, partial least squares regression,
ST:STUDY_SUMMARY                 	univariate linear modeling and multiple hypothesis testing. With these results,
ST:STUDY_SUMMARY                 	we extract association insights that reveal the most influential metabolites per
ST:STUDY_SUMMARY                 	each phenotypic feature.
ST:INSTITUTE                     	Purdue University
ST:DEPARTMENT                    	Biomedical Engineering-Veterinary Clinical Sciences
ST:LAST_NAME                     	Munoz-Briones
ST:FIRST_NAME                    	Javier
ST:ADDRESS                       	610 Purdue Mall, West Lafayette, IN 47907
ST:EMAIL                         	munoz99@purdue.edu
ST:PHONE                         	7657469102
ST:TOTAL_SUBJECTS                	16
ST:NUM_MALES                     	9
ST:NUM_FEMALES                   	7
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Ovis aries
SU:TAXONOMY_ID                   	9940
SU:AGE_OR_AGE_RANGE              	8-61
SU:WEIGHT_OR_WEIGHT_RANGE        	44-95
SU:GENDER                        	Male and female
SU:ANIMAL_ANIMAL_SUPPLIER        	West Lafayette, Indiana, United States
SU:ANIMAL_HOUSING                	West Lafayette, Indiana, United States
SU:ANIMAL_FEED                   	Aad libitum hay (75% grass, 25% alfalfa)
SU:ANIMAL_WATER                  	Free access to water
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	PURD-00075	B1S5	Sex:Female | Breed:Hampshire	Tag=1981; Weight=73; Age=37
SUBJECT_SAMPLE_FACTORS           	PURD-00078	B1S9	Sex:Female | Breed:Cross.bred	Tag=1662; Weight=75; Age=61
SUBJECT_SAMPLE_FACTORS           	PURD-00081	B1S12	Sex:Female | Breed:Dorset	Tag=13402; Weight=53; Age=49
SUBJECT_SAMPLE_FACTORS           	PURD-00084	B1S13	Sex:Female | Breed:Cross.bred	Tag=1822; Weight=64; Age=61
SUBJECT_SAMPLE_FACTORS           	PURD-00087	B3S2	Sex:Male | Breed:Cross.bred	Tag=1194; Weight=66; Age=8
SUBJECT_SAMPLE_FACTORS           	PURD-00090	B3S4	Sex:Male | Breed:Cross.bred	Tag=1117; Weight=44; Age=25
SUBJECT_SAMPLE_FACTORS           	PURD-00093	B3S6	Sex:Male | Breed:Cross.bred	Tag=1107; Weight=57; Age=8
SUBJECT_SAMPLE_FACTORS           	PURD-00096	B3S8	Sex:Male | Breed:Cross.bred	Tag=20241; Weight=95; Age=37
SUBJECT_SAMPLE_FACTORS           	PURD-00117	B3S7	Sex:Male | Breed:Cross.bred	Tag=1110; Weight=52; Age=25
SUBJECT_SAMPLE_FACTORS           	PURD-00120	B3S9	Sex:Male | Breed:Cross.bred	Tag=LO406; Weight=90; Age=49
SUBJECT_SAMPLE_FACTORS           	PURD-00099	B1S2	Sex:Female | Breed:Hampshire	Tag=1958; Weight=64; Age=37
SUBJECT_SAMPLE_FACTORS           	PURD-00102	B1S8	Sex:Female | Breed:Cross.bred	Tag=2476; Weight=66; Age=25
SUBJECT_SAMPLE_FACTORS           	PURD-00105	B1S10	Sex:Female | Breed:Ramboilette	Tag=2476; Weight=67; Age=61
SUBJECT_SAMPLE_FACTORS           	PURD-00108	B3S1	Sex:Male | Breed:Cross.bred	Tag=1154; Weight=40; Age=49
SUBJECT_SAMPLE_FACTORS           	PURD-00111	B3S3	Sex:Male | Breed:Dorset	Tag=1103; Weight=58; Age=8
SUBJECT_SAMPLE_FACTORS           	PURD-00114	B3S5	Sex:Male | Breed:Dorset	Tag=1112; Weight=47; Age=49
#COLLECTION
CO:COLLECTION_SUMMARY            	Rumenomics. Rumen fluid was collected post morten in a necropsy laboratory from
CO:COLLECTION_SUMMARY            	rumen. Samples were filtered using cheese cloth and were transported at 4°C at
CO:COLLECTION_SUMMARY            	the laboratory to be preprocessed. Samples were centrifuged at 25000 rpm for 15
CO:COLLECTION_SUMMARY            	min, then syringe filters 0.45 um pore size were used to remove solids and
CO:COLLECTION_SUMMARY            	storage at 20 °C while they were sent to Metabolon.
CO:SAMPLE_TYPE                   	Sheep Rumen Fluid
CO:COLLECTION_LOCATION           	West Lafayette, Indiana, United States
CO:COLLECTION_FREQUENCY          	One-time
CO:COLLECTION_DURATION           	10 minutes
CO:COLLECTION_TUBE_TEMP          	-80°C
#TREATMENT
TR:TREATMENT_SUMMARY             	Sheep were healthy by their age and weight. They were not under treatment
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	: Samples were prepared using the automated MicroLab STAR® system from Hamilton
SP:SAMPLEPREP_SUMMARY            	Company. Several recovery standards were added prior to the first step in the
SP:SAMPLEPREP_SUMMARY            	extraction process for QC purposes. To remove protein, dissociate small
SP:SAMPLEPREP_SUMMARY            	molecules bound to protein or trapped in the precipitated protein matrix, and to
SP:SAMPLEPREP_SUMMARY            	recover chemically diverse metabolites, proteins were precipitated with methanol
SP:SAMPLEPREP_SUMMARY            	under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by
SP:SAMPLEPREP_SUMMARY            	centrifugation. The resulting extract was divided into five fractions: two for
SP:SAMPLEPREP_SUMMARY            	analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion
SP:SAMPLEPREP_SUMMARY            	mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with
SP:SAMPLEPREP_SUMMARY            	negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion
SP:SAMPLEPREP_SUMMARY            	mode ESI, and one sample was reserved for backup. Samples were placed briefly on
SP:SAMPLEPREP_SUMMARY            	a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were
SP:SAMPLEPREP_SUMMARY            	stored overnight under nitrogen before preparation for analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Low pH Lipophilic (LC/MS Pos late)
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters Acquity BEH C18 (100 x 2mm, 1.7um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5
CH:SOLVENT_B                     	50% methanol/50% acetonitrile; 0.1% formic acid; 0.05% PFPA, pH ~2.5
CH:FLOW_GRADIENT                 	Linear gradient from 40% B to 99.5% B over 1.0 minute, hold 99.5% B for 2.4
CH:FLOW_GRADIENT                 	minutes.
CH:FLOW_RATE                     	0.60 mL/min
CH:COLUMN_TEMPERATURE            	40-50
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	Other
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Metabolon (LC/MS Pos late)
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Batch normalized peak area
MS_METABOLITE_DATA_START
Samples	B1S5	B1S9	B1S12	B1S13	B3S2	B3S4	B3S6	B3S8	B3S7	B3S9	B1S2	B1S8	B1S10	B3S1	B3S3	B3S5
Factors	Sex:Female | Breed:Hampshire	Sex:Female | Breed:Cross.bred	Sex:Female | Breed:Dorset	Sex:Female | Breed:Cross.bred	Sex:Male | Breed:Cross.bred	Sex:Male | Breed:Cross.bred	Sex:Male | Breed:Cross.bred	Sex:Male | Breed:Cross.bred	Sex:Male | Breed:Cross.bred	Sex:Male | Breed:Cross.bred	Sex:Female | Breed:Hampshire	Sex:Female | Breed:Cross.bred	Sex:Female | Breed:Ramboilette	Sex:Male | Breed:Cross.bred	Sex:Male | Breed:Dorset	Sex:Male | Breed:Dorset
sphinganine	0.849606591	1.706926416	0.347708539	0.738215161	1.717369294	1.424969234	2.436168131	0.918634933	1.856716855	1.121159699	0.765444039	1.054350295	0.493241287	0.559546497	1.896119639	0.875433062
cortisol	0.930894486	0.697415346	1.654961607	0.71537141	0.268039443	1.17012539	1.38096443	0.94243471	1.246710625	1.40899052	0.677354011	1.101239754	0.476922954	0.412863995	1.072895239	1.048954807
phytosphingosine	0.819786023	1.178626398	0.458337905	0.514257075	1.877722783	1.608476843	1.478783673	0.578464081	1.283149615	0.575368005	0.697429723	0.8534	0.518088618	0.906990193	0.982848014	0.999971333
alpha-tocopherol	0.681924402	1.546251716	0.4908744	0.374767798	1.105954556	1.816026556	2.075615415	0.411826046	0.877491561	0.529706368	0.954052543	1.037200021	0.569164967	3.031227666	1.503803608	3.681714346
pheophorbide A	0.295759817	1.49193554	0.192983719	0.321147616	1.086516261	3.240480366	2.214492609	0.099383219	0.516944576	0.203073412	0.63853325	1.497486737	0.725589507	2.98449669	1.472809494	3.408041641
pheophytin A	0.391806879	1.596430943	0.247025432	0.314989089	1.251991663	2.157815551	3.424482476	0.092121044	0.371764799	0.078766032	1.150206498	0.842721658	0.678585884	6.316707342	1.396344316	3.933801197
4-cholesten-3-one	0.960040147	1.462063708	0.412466683	0.821590107	0.629091855	2.414844468	1.27503673	0.403608612	0.608492276	0.403608612	0.473173916	0.422034347	0.484302073	1.107230629	1.384372077	1.253732905
gamma-tocopherol/beta-tocopherol	0.831732779	1.675517058	0.398414907	0.35839145	0.535518211	1.085861788	1.43870276	0.35839145	0.43054492	0.35839145	1.043338008	1.021718864	0.442561655	2.194699463	0.697933963	1.63613858
N-palmitoyl-sphinganine (d18:0/16:0)	1.505336588	1.772732358	0.514148056	0.982645214	1.310627647	1.233050709	1.127783314	0.612178591	1.233727421	0.671218784	1.013609508	1.787732984	0.917363754	0.240329392	0.76660223	0.885693354
carotene diol (1)	0.602305205	1.541333131	0.443831688	0.242849096	1.164417674	2.600647866	2.066580925	0.188490091	0.611906356	0.259016933	1.112003089	0.866129299	0.708476024	4.235447935	1.324711616	3.451524109
carotene diol (2)	0.479372842	1.157103353	0.352428776	0.225892721	0.934844128	2.676697672	2.251347989	0.125019097	0.429209647	0.143925754	1.119044636	0.518283595	0.558732096	3.474916082	1.193329914	2.851765988
carotene diol (3)	0.26965279	0.344073464	0.26965279	0.26965279	0.355111972	1.297803662	1.123053306	0.26965279	0.296014862	0.26965279	0.26965279	0.26965279	0.26965279	1.817174385	0.548888855	1.319812499
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	SUPER_PATHWAY	SUB_PATHWAY	TYPE	INCHIKEY	SMILES	CAS	CHEMSPIDER	HMDB	KEGG	PUBCHEM
sphinganine	Lipid	Sphingolipid Synthesis	NAMED	OTKJDMGTUTTYMP-UHFFFAOYSA-N	CCCCCCCCCCCCCCCC(C(CO)N)O	3102-56-5	3014	HMDB0000269	C00836	3126
cortisol	Lipid	Corticosteroids	NAMED	JYGXADMDTFJGBT-VWUMJDOOSA-N	O[C@@]1([C@@](CO)=O)[C@]2(C)[C@](CC1)([H])[C@@](CCC3=CC4=O)([H])[C@]([C@@]3(C)CC4)([H])[C@@H](O)C2	50-23-7		HMDB0000063	C00735	5754
phytosphingosine	Lipid	Sphingolipid Synthesis	NAMED	AERBNCYCJBRYDG-KSZLIROESA-N	O[C@@H]([C@@H](O)[C@@H](N)CO)CCCCCCCCCCCCCC	554-62-1	21238683	HMDB0004610	C12144	122121
alpha-tocopherol	Cofactors and Vitamins	Tocopherol Metabolism	NAMED	GVJHHUAWPYXKBD-IEOSBIPESA-N	CC1=C2C(O[C@@](CCC[C@H](C)CCC[C@H](C)CCC[C@H](C)C)(C)CC2)=C(C)C(C)=C1O	10191-41-0,59-02-9	14265	HMDB0001893	C02477	86472
pheophorbide A	Xenobiotics	Food Component/Plant	NAMED	NSFSLUUZQIAOOX-UHFFFAOYSA-N	CC(/C(N1)=C/C(C(CC)=C/2C)=NC2=C/C(N3)=C4C=C)=C(C(C5C(OC)=O)=O)C1=C5C(C(C/6C)CCC(O)=O)=NC6=C/C3=C4C	15664-29-6			C18021	253193
pheophytin A	Xenobiotics	Food Component/Plant	NAMED	CQIKWXUXPNUNDV-HOIIHFJFSA-N	O=C1[C@H]([C@](OC)=O)C(C([C@@H](CCC(OC/C=C(C)/CCC[C@@H](C)CCC[C@@H](C)CCCC(C)C)=O)[C@@H]/2C)=NC2=C/C3=C(C)C(C=C)=C4N3)=C(N/C5=CC(C(CC)=C/6C)=NC6=C/4)C1=C5C	603-17-8			C05797	135398712
4-cholesten-3-one	Lipid	Sterol	NAMED	NYOXRYYXRWJDKP-UHFFFAOYSA-N	CC12C(CCC3C2CCC4(C)C3CCC4C(CCCC(C)C)C)=CC(CC1)=O	601-57-0	427	HMDB0000921	C00599	91477
gamma-tocopherol/beta-tocopherol	Cofactors and Vitamins	Tocopherol Metabolism	NAMED	QUEDXNHFTDJVIY-DQCZWYHMSA-N	CC1=C(C(CC2)=CC(O)=C1C)O[C@@]2(CCC[C@@H](CCC[C@@H](CCCC(C)C)C)C)C			HMDB0006335,HMDB0001492	C14152,C02483
N-palmitoyl-sphinganine (d18:0/16:0)	Lipid	Dihydroceramides	NAMED	GCGTXOVNNFGTPQ-JHOUSYSJSA-N	[H][C@](NC(CCCCCCCCCCCCCCC)=O)(CO)[C@@](O)([H])CCCCCCCCCCCCCCC	5966-29-0	4446685	HMDB11760		5283572
carotene diol (1)	Cofactors and Vitamins	Vitamin A Metabolism	NAMED	JKQXZKUSFCKOGQ-DKLMTRRASA-N	CC1=C(/C=C/C(C)=C/C=C/C(C)=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C2=C(C)CC(O)CC2(C)C)C(C)(C)CC(O)C1
carotene diol (2)	Cofactors and Vitamins	Vitamin A Metabolism	NAMED	JKQXZKUSFCKOGQ-DKLMTRRASA-N	CC1=C(/C=C/C(C)=C/C=C/C(C)=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C2=C(C)CC(O)CC2(C)C)C(C)(C)CC(O)C1
carotene diol (3)	Cofactors and Vitamins	Vitamin A Metabolism	NAMED	JKQXZKUSFCKOGQ-DKLMTRRASA-N	CC1=C(/C=C/C(C)=C/C=C/C(C)=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C2=C(C)CC(O)CC2(C)C)C(C)(C)CC(O)C1
METABOLITES_END
#END