#METABOLOMICS WORKBENCH ebrown427_20150608_9259991_mwtab.txt DATATRACK_ID:291 STUDY_ID:ST000222 ANALYSIS_ID:AN000331 PROJECT_ID:PR000182 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Metabolomic analysis of the small intestinal content of malnourished mice PR:PROJECT_SUMMARY - PR:INSTITUTE University of British Columbia PR:DEPARTMENT Microbiology and Immunology PR:LABORATORY Dr. B. Brett Finlay PR:LAST_NAME Finlay PR:FIRST_NAME Brett PR:ADDRESS 2185 East Mall, Vancouver, BC, Canada V6T 1Z4 PR:EMAIL bfinlay@msl.ubc.ca PR:PHONE - PR:FUNDING_SOURCE Canadian Institutes for Health Research #STUDY ST:STUDY_TITLE Bile acid targeted metabolomics of the small intestine in malnourished and ST:STUDY_TITLE mice ST:STUDY_TYPE Targeted metabolomics ST:STUDY_SUMMARY A total of 8 samples from 6 week old, female C57BL/6 mice, treated for 3 weeks ST:STUDY_SUMMARY a malnourished diet or a control-fed isocaloric diet. Samples were taken from ST:STUDY_SUMMARY small intestinal fecal content at the terminus of the ileum for targeted bile ST:STUDY_SUMMARY analysis. ST:INSTITUTE University of Victoria ST:DEPARTMENT The Uvic Proteomics and Metabolomics Innovation Centre ST:LAST_NAME Borchers ST:FIRST_NAME Christoph ST:ADDRESS #3101-4464 Markham St Victoria, BC Canada, V8Z 7X8 ST:EMAIL christoph@proteincentre.com ST:PHONE - ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 8* ST:STUDY_COMMENTS *One sample failed quality control in the malnourished group, thus was not ST:STUDY_COMMENTS in the study #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:AGE_OR_AGE_RANGE 6 weeks at collection SU:GENDER Female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs SU:ANIMAL_HOUSING Conventional SU:ANIMAL_FEED Low Protein, Low Fat Malnourished Diet/Isocaloric Control Diet SU:CELL_PRIMARY_IMMORTALIZED Malnourished/Control #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Control_SI_1 CON1 Diet:control SUBJECT_SAMPLE_FACTORS Control_SI_2 CON2 Diet:control SUBJECT_SAMPLE_FACTORS Control_SI_3 CON3 Diet:control SUBJECT_SAMPLE_FACTORS Control_SI_4 CON4 Diet:control SUBJECT_SAMPLE_FACTORS Malnourished_SI_1 MAL1 Diet:malnourished SUBJECT_SAMPLE_FACTORS Malnourished_SI_2 MAL2 Diet:malnourished SUBJECT_SAMPLE_FACTORS Malnourished_SI_3 MAL3 Diet:malnourished SUBJECT_SAMPLE_FACTORS Malnourished_SI_4 MAL4 Diet:malnourished #COLLECTION CO:COLLECTION_SUMMARY - CO:SAMPLE_TYPE Small intestinal fecal contents CO:COLLECTION_TIME 6 weeks of age CO:STORAGE_CONDITIONS -80 degrees CO:TISSUE_CELL_QUANTITY_TAKEN 15-40mg #TREATMENT TR:TREATMENT_SUMMARY 4 mice were given a malnourished diet and 4 mice were untreated, fed a control #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Bile-acid targeted metabolomics. Each sample was homogenized in LC-MS grade SP:SAMPLEPREP_SUMMARY at a ratio of 150 ?L per 10 mg raw material and with the aid of 5-mm stainless SP:SAMPLEPREP_SUMMARY metal balls. Bile acids were extracted by addition of acetonitrile at a ratio SP:SAMPLEPREP_SUMMARY 350 ?L per 10 mg raw material followed by vortexing and sonication (1 min) in SP:SAMPLEPREP_SUMMARY ice-water ultrasonic bath. The samples were centrifuged. 20 ?L of the SP:SAMPLEPREP_SUMMARY were precisely taken out and mixed with a predefined mix of 14 SP:SAMPLEPREP_SUMMARY bile acids as the internal standards. The mixtures were subjected to SP:SAMPLEPREP_SUMMARY solid-phase extraction according to a validated protocol for sample cleanup and SP:SAMPLEPREP_SUMMARY acid enrichment62. The flow-through fractions were collected and then dried SP:SAMPLEPREP_SUMMARY a gentle nitrogen flow. The dried residues were dissolved in 200 ?L of 50% SP:SAMPLEPREP_SUMMARY 10 ?L were injected for quantitation by UPLC-MRM/MS. A Dionex UPLC system was SP:SAMPLEPREP_SUMMARY to an AB Sciex 4000 QTRAP mass spectrometer which was operated in the negative SP:SAMPLEPREP_SUMMARY multiple-reaction monitoring (MRM) mode and with electrospray ionization. UPLC SP:SAMPLEPREP_SUMMARY was carried out on a 15 cm long C-18 UPLC column with water-acetonitrile-formic SP:SAMPLEPREP_SUMMARY as the mobile phase for binary gradient elution using a developed and validated SP:SAMPLEPREP_SUMMARY for comprehensive analysis of bile acids in biological samples (Han, etc. SP:SAMPLEPREP_SUMMARY submitted to Analytical Chemistry). The column temperature was 45 oC and the SP:SAMPLEPREP_SUMMARY rate was 0.35 mL/min. 45 bile acids (including the 19 targeted bile acids) were SP:SAMPLEPREP_SUMMARY in the quantitation by UPLC/scheduled MRM/MS. Concentrations of the detected SP:SAMPLEPREP_SUMMARY acids were calculated with internal standard calibration from the linearly SP:SAMPLEPREP_SUMMARY standard calibration curves of individual bile acids. The lower limits of SP:SAMPLEPREP_SUMMARY were 0.08 nmoles/mg for all the bile acids. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex API 4000 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nM/mg MS_METABOLITE_DATA_START Samples CON1 CON2 CON3 CON4 MAL1 MAL2 MAL3 MAL4 Factors Diet:control Diet:control Diet:control Diet:control Diet:malnourished Diet:malnourished Diet:malnourished Diet:malnourished 12-Ketochenodeoxycholic acid 108.7730 1.3820 7.8860 7.2030 12.4300 4.6520 0.0000 0.8890 3-Oxocholic acid 6.8920 0.1110 1.0330 0.8540 0.7920 0.4250 0.0000 1.1570 7-12-Ketolithocholic acid 2.1870 0.0000 0.3590 0.1960 0.8320 0.2250 0.0000 0.5700 7-Ketodeoxycholic acid 3.0704 0.7200 6.6340 4.9730 16.0190 4.7440 0.0000 11.8960 7-Ketolithocholic acid 1.7674 0.1590 1.4570 1.6570 1.5330 1.2340 0.0000 0.0850 Allocholic acid 1.4670 0.0180 0.1140 0.0900 0.2640 0.0870 0.0000 0.0550 a-Muricholic acid 7.8133 4.5970 0.2560 15.7920 37.2710 11.5280 0.3440 3.2230 b-Muricholic acid 72.0297 18.6790 124.0930 83.3740 634.4700 186.3680 2.0130 83.3590 Chenodeoxycholic acid 6.3277 1.0050 6.7610 4.1130 9.1510 8.3550 0.1200 1.0680 Cholic acid 1960.5300 47.6040 357.6140 223.0970 856.6850 299.6020 1.4750 164.7670 Dehydrolithocholic acid 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 Deoxycholic acid 6.4202 0.9840 12.1270 6.0950 38.2260 14.0890 0.2460 18.0430 Dioxolithocholic acid 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 Glycochenodeoxycholic acid 0.0000 0.0000 0.1010 0.0000 0.0000 0.1080 0.0000 0.0000 Glycocholic acid 1.4330 2.1090 5.3160 3.4020 2.4370 3.5920 0.0000 0.4310 Hyodeoxycholic acid 4.0018 0.3030 2.8260 1.3760 2.4980 0.2660 0.0230 1.3350 Isolithocholic acid 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 lam-MCA hyocholic acid 4.0300 0.0000 0.3440 0.3060 0.4480 0.2650 0.0000 0.1110 Lithocholic acid 0.6050 0.0520 0.1020 0.0730 0.0910 0.0860 0.0380 0.0580 Murocholic acid 0.1995 0.1240 1.2120 0.6630 4.0050 1.1300 0.0000 0.8530 Tauro-a-muricholic acid 90.8230 153.7910 272.9110 294.7870 191.3270 99.0960 1.0090 22.6630 Tauro-b-muricholic acid 679.5520 775.2880 1643.5810 573.3680 1284.4940 650.6100 7.6530 379.7440 Taurochenodexycholic acid 29.6820 35.0870 61.6400 72.7730 21.2490 20.1210 0.0940 2.5490 Taurocholic acid 193.7090 690.7660 1546.0760 1219.1530 1020.8630 504.3730 5.8910 174.9120 Taurodeoxycholic acid 23.6950 25.2320 76.8910 49.1540 58.3880 23.5710 0.1770 28.7400 Taurohyocholic acid (tauro-?-MCA) 0.3360 0.6690 0.7420 0.7120 0.6280 0.2930 0.0000 0.2790 Taurohyodeoxycholic acid 63.5980 45.9080 108.4530 89.4540 36.3660 17.7530 0.1610 10.7030 Taurolithocholic acid 1.5680 0.4370 1.0410 0.9630 0.5470 0.2340 0.0000 Tauroursodexycholic acid 63.6590 46.8690 114.4200 97.8610 36.2920 18.6230 0.2460 11.0130 Tauro-w-muricholic acid 96.6590 163.4190 232.1770 224.7020 91.0140 34.8140 0.4930 84.7320 Ursocholic acid 1.1780 0.0120 0.5250 0.3480 2.5870 1.3010 0.0000 0.5180 Ursodeoxycholic acid 6.1289 0.4410 5.2620 3.2880 27.0110 21.1920 0.0000 2.7360 w-Muricholic acid 4.2596 1.4930 5.4190 4.4960 7.2730 2.7010 0.0000 7.9960 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name moverz_quant ri ri_type pubchem_id inchi_key kegg_id other_id other_id_type 12-Ketochenodeoxycholic acid Uvictoria_ID 3-Oxocholic acid Uvictoria_ID 7-12-Ketolithocholic acid Uvictoria_ID 7-Ketodeoxycholic acid Uvictoria_ID 7-Ketolithocholic acid Uvictoria_ID Allocholic acid Uvictoria_ID a-Muricholic acid Uvictoria_ID b-Muricholic acid Uvictoria_ID Chenodeoxycholic acid Uvictoria_ID Cholic acid Uvictoria_ID Dehydrolithocholic acid Uvictoria_ID Deoxycholic acid Uvictoria_ID Dioxolithocholic acid Uvictoria_ID Glycochenodeoxycholic acid Uvictoria_ID Glycocholic acid Uvictoria_ID Hyodeoxycholic acid Uvictoria_ID Isolithocholic acid Uvictoria_ID lam-MCA hyocholic acid Uvictoria_ID Lithocholic acid Uvictoria_ID Murocholic acid Uvictoria_ID Tauro-a-muricholic acid Uvictoria_ID Tauro-b-muricholic acid Uvictoria_ID Taurochenodexycholic acid Uvictoria_ID Taurocholic acid Uvictoria_ID Taurodeoxycholic acid Uvictoria_ID Taurohyocholic acid (tauro-?-MCA) Uvictoria_ID Taurohyodeoxycholic acid Uvictoria_ID Taurolithocholic acid Uvictoria_ID Tauroursodexycholic acid Uvictoria_ID Tauro-w-muricholic acid Uvictoria_ID Ursocholic acid Uvictoria_ID Ursodeoxycholic acid Uvictoria_ID w-Muricholic acid Uvictoria_ID METABOLITES_END #END