#METABOLOMICS WORKBENCH ryant_20200402_080758 DATATRACK_ID:1976 STUDY_ID:ST001361 ANALYSIS_ID:AN002265 PROJECT_ID:PR000931 VERSION 1 CREATED_ON April 16, 2020, 5:48 am #PROJECT PR:PROJECT_TITLE Tryptophan metabolomics in CKD serum PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Targeted tryptophan metabolomics were performed in mouse serum collected from PR:PROJECT_SUMMARY mice with and without chronic kidney disease PR:INSTITUTE University of Florida PR:DEPARTMENT Applied Physiolog and Kinesiology PR:LAST_NAME Ryan PR:FIRST_NAME Terence PR:ADDRESS 1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA PR:EMAIL ryant@ufl.edu PR:PHONE 352-294-1700 PR:FUNDING_SOURCE NIH/NHLBIR01-HL149704 and R01-HL148597 #STUDY ST:STUDY_TITLE Serum tryptophan metabolomics in CKD ST:STUDY_TYPE MS quantitative analysis ST:STUDY_SUMMARY Serum was processed using a targeted metabolomics platform for quantifying ST:STUDY_SUMMARY tryptophan metabolites as a number of these metabolites are well establish ST:STUDY_SUMMARY uremic toxins. ST:INSTITUTE University of Florida ST:LAST_NAME Ryan ST:FIRST_NAME Terence ST:ADDRESS 1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA ST:EMAIL ryant@ufl.edu ST:PHONE 352-294-1700 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 16 ST:NUM_MALES 8 ST:NUM_FEMALES 8 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL6J SU:AGE_OR_AGE_RANGE 18-20 weeks SU:WEIGHT_OR_WEIGHT_RANGE 20-30g SU:GENDER Male and female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs SU:ANIMAL_HOUSING 5/cage SU:ANIMAL_LIGHT_CYCLE 12h SU:ANIMAL_FEED Ad libitum. Control mice received custom casein-diet. Chronic kidney disease was SU:ANIMAL_FEED induced by supplementing casein-based diet with 0.15% adenine SU:ANIMAL_WATER Ad libitum #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 Std Bracket Sample C1 Group:N/A Sex=N/A; RAW_FILE_NAME=C1.raw SUBJECT_SAMPLE_FACTORS Std Bracket Sample C2 Group:N/A Sex=N/A; RAW_FILE_NAME=C2.raw SUBJECT_SAMPLE_FACTORS Std Bracket Sample C3 Group:N/A Sex=N/A; RAW_FILE_NAME=C3.raw SUBJECT_SAMPLE_FACTORS Std Bracket Sample C4 Group:N/A Sex=N/A; RAW_FILE_NAME=C4.raw SUBJECT_SAMPLE_FACTORS Std Bracket Sample C5 Group:N/A Sex=N/A; RAW_FILE_NAME=C5.raw SUBJECT_SAMPLE_FACTORS Std Bracket Sample C6 Group:N/A Sex=N/A; RAW_FILE_NAME=C6.raw SUBJECT_SAMPLE_FACTORS Std Bracket Sample C7 Group:N/A Sex=N/A; RAW_FILE_NAME=C7.raw SUBJECT_SAMPLE_FACTORS QC Sample QC1 Group:N/A Sex=N/A; RAW_FILE_NAME=QC1.raw SUBJECT_SAMPLE_FACTORS QC Sample QC2 Group:N/A Sex=N/A; RAW_FILE_NAME=QC2.raw SUBJECT_SAMPLE_FACTORS QC Sample QC3 Group:N/A Sex=N/A; RAW_FILE_NAME=QC3.raw SUBJECT_SAMPLE_FACTORS mouse serum CKD001 Group:CKD Sex=Male; RAW_FILE_NAME=CKD001.raw SUBJECT_SAMPLE_FACTORS mouse serum CKD002 Group:CKD Sex=Male; RAW_FILE_NAME=CKD002.raw SUBJECT_SAMPLE_FACTORS mouse serum CKD003 Group:CKD Sex=Male; RAW_FILE_NAME=CKD003.raw SUBJECT_SAMPLE_FACTORS mouse serum CKD004 Group:CKD Sex=Male; RAW_FILE_NAME=CKD004.raw SUBJECT_SAMPLE_FACTORS mouse serum CKD005 Group:CKD Sex=Female; RAW_FILE_NAME=CKD005.raw SUBJECT_SAMPLE_FACTORS mouse serum CKD006 Group:CKD Sex=Female; RAW_FILE_NAME=CKD006.raw SUBJECT_SAMPLE_FACTORS mouse serum CKD007 Group:CKD Sex=Female; RAW_FILE_NAME=CKD007.raw SUBJECT_SAMPLE_FACTORS mouse serum CKD008 Group:CKD Sex=Female; RAW_FILE_NAME=CKD008.raw SUBJECT_SAMPLE_FACTORS mouse serum Con001 Group:Control Sex=Male; RAW_FILE_NAME=Con001.raw SUBJECT_SAMPLE_FACTORS mouse serum Con002 Group:Control Sex=Male; RAW_FILE_NAME=Con002.raw SUBJECT_SAMPLE_FACTORS mouse serum Con003 Group:Control Sex=Male; RAW_FILE_NAME=Con003.raw SUBJECT_SAMPLE_FACTORS mouse serum Con004 Group:Control Sex=Male; RAW_FILE_NAME=Con004.raw SUBJECT_SAMPLE_FACTORS mouse serum Con005 Group:Control Sex=Female; RAW_FILE_NAME=Con005.raw SUBJECT_SAMPLE_FACTORS mouse serum Con006 Group:Control Sex=Female; RAW_FILE_NAME=Con006.raw SUBJECT_SAMPLE_FACTORS mouse serum Con007 Group:Control Sex=Female; RAW_FILE_NAME=Con007.raw SUBJECT_SAMPLE_FACTORS mouse serum Con008 Group:Control Sex=Female; RAW_FILE_NAME=Con008.raw #COLLECTION CO:COLLECTION_SUMMARY Serum was collected ketamine/xylazine anesthesia, from a 1mm tail snip, allowed CO:COLLECTION_SUMMARY to clot for 20 minutes at room temperature and centrifuged at 4000xG for 10 min. CO:COLLECTION_SUMMARY Serum was collected from stored at -80C until analysis. CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY We utilized an established adenine-diet model to induce CKD in mice. Mice were TR:TREATMENT_SUMMARY assigned to a casein-based chow diet for 7 days, followed by induction of renal TR:TREATMENT_SUMMARY tubular injury by supplementing the diet with 0.2% adenine for 7 days, and were TR:TREATMENT_SUMMARY subsequently maintained on a 0.15% adenine diet for 7 more weeks. CKD mice were TR:TREATMENT_SUMMARY then placed back on control casein diet for 2 weeks to prior to euthanasia and TR:TREATMENT_SUMMARY terminal experiments. Control mice received casein diet for the duration of the TR:TREATMENT_SUMMARY study. TR:ANIMAL_ANESTHESIA Ketamine/Xylazine TR:ANIMAL_ENDP_EUTHANASIA Ketamine/Xylazine #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Twenty-five microliters of each mouse serum was spiked with 5 µL internal SP:SAMPLEPREP_SUMMARY standards (IS) solution consisted of tryptophan ¹³C₁₁, serotonin D4, SP:SAMPLEPREP_SUMMARY kynurenine D4, kynurenic Acid D5, xanthurenic acid D4, anthranilic acid SP:SAMPLEPREP_SUMMARY ¹³C₆, indoxyl sulfate ¹³C₆, p-cresol sulfate D7 and 3-indole-acetate D7. SP:SAMPLEPREP_SUMMARY Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 SP:SAMPLEPREP_SUMMARY Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein SP:SAMPLEPREP_SUMMARY precipitation was allowed by incubating the samples at 4°C for 20 min. Samples SP:SAMPLEPREP_SUMMARY were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg SP:SAMPLEPREP_SUMMARY for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from SP:SAMPLEPREP_SUMMARY each sample into clean tube and dried under a gentle stream of nitrogen at SP:SAMPLEPREP_SUMMARY 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic SP:SAMPLEPREP_SUMMARY acid. Resuspension was allowed at 4°C for 10 -15 min then samples were SP:SAMPLEPREP_SUMMARY centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into SP:SAMPLEPREP_SUMMARY clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap SP:SAMPLEPREP_SUMMARY mass spectrometer with Dionex UHPLC and autosampler. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Twenty-five microliters of each mouse serum was spiked with 5 µL internal CH:CHROMATOGRAPHY_SUMMARY standards (IS) solution consisted of tryptophan ¹³C₁₁, serotonin D4, CH:CHROMATOGRAPHY_SUMMARY kynurenine D4, kynurenic Acid D5, xanthurenic acid D4, anthranilic acid CH:CHROMATOGRAPHY_SUMMARY ¹³C₆, indoxyl sulfate ¹³C₆, p-cresol sulfate D7 and 3-indole-acetate D7. CH:CHROMATOGRAPHY_SUMMARY Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 CH:CHROMATOGRAPHY_SUMMARY Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein CH:CHROMATOGRAPHY_SUMMARY precipitation was allowed by incubating the samples at 4°C for 20 min. Samples CH:CHROMATOGRAPHY_SUMMARY were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg CH:CHROMATOGRAPHY_SUMMARY for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from CH:CHROMATOGRAPHY_SUMMARY each sample into clean tube and dried under a gentle stream of nitrogen at CH:CHROMATOGRAPHY_SUMMARY 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic CH:CHROMATOGRAPHY_SUMMARY acid. Resuspension was allowed at 4°C for 10 -15 min then samples were CH:CHROMATOGRAPHY_SUMMARY centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into CH:CHROMATOGRAPHY_SUMMARY clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap CH:CHROMATOGRAPHY_SUMMARY mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed CH:CHROMATOGRAPHY_SUMMARY in positive and negative heated electrospray ionization for all with a mass CH:CHROMATOGRAPHY_SUMMARY resolution of 35,000 at m/z 200 as separate injections. Tryptophan, serotonin, CH:CHROMATOGRAPHY_SUMMARY kynurenine, kynurenic acid, xanthurenic acid and anthranilic acid were CH:CHROMATOGRAPHY_SUMMARY quantified in the positive ionization while indoxyl sulfate, p-cresol sulfate CH:CHROMATOGRAPHY_SUMMARY and 3-indole-acetate were analyzed in negative ionization. Separation was CH:CHROMATOGRAPHY_SUMMARY achieved on an ACE 18-PFP 100 x 2.1 mm, 2 µm column using a gradient with CH:CHROMATOGRAPHY_SUMMARY mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. CH:CHROMATOGRAPHY_SUMMARY The flow rate was 350 µL/min with a column temperature of 25°C and injection CH:CHROMATOGRAPHY_SUMMARY volume of 2 µL. Run time was 20.5 min. A 9-point calibration curve and QC CH:CHROMATOGRAPHY_SUMMARY samples were prepared for targeted quantitation of tryptophan, serotonin, CH:CHROMATOGRAPHY_SUMMARY kynurenine, kynurenic acid, xanthurenic acid, anthranilic acid, indoxyl sulfate, CH:CHROMATOGRAPHY_SUMMARY p-cresol sulfate and 3-indole-acetate. 20 µL of each calibrator and QCs were CH:CHROMATOGRAPHY_SUMMARY supplemented with 5 µl indoxyl sulfate. Peak areas of each analyte and CH:CHROMATOGRAPHY_SUMMARY corresponding internal standard in the calibrator, QCs and samples were CH:CHROMATOGRAPHY_SUMMARY integrated using Xcalibur 4.0. A calibration curve was generated by plotting CH:CHROMATOGRAPHY_SUMMARY nominal concentration of the analyte in the calibrators versus peak area ratio CH:CHROMATOGRAPHY_SUMMARY of analyte and IS. QCs and samples were quantitated against the calibration CH:CHROMATOGRAPHY_SUMMARY curve. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex CH:COLUMN_NAME ACE 5 C18-300 (100 x 2.1mm) CH:FLOW_RATE 350ul/min CH:COLUMN_TEMPERATURE 25C CH:SOLVENT_A 0.1% formic acid CH:SOLVENT_B acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Tryptophan, serotonin, kynurenine, kynurenic acid, xanthurenic acid and MS:MS_COMMENTS anthranilic acid were quantified in the positive ionization while indoxyl MS:MS_COMMENTS sulfate, p-cresol sulfate and 3-indole-acetate were analyzed in negative MS:MS_COMMENTS ionization. Separation was achieved on an ACE 18-PFP 100 x 2.1 mm, 2 µm column MS:MS_COMMENTS using a gradient with mobile phase A as 0.1% formic acid in water and mobile MS:MS_COMMENTS phase B as acetonitrile. The flow rate was 350 µL/min with a column temperature MS:MS_COMMENTS of 25°C and injection volume of 2 µL. Run time was 20.5 min. A 9-point MS:MS_COMMENTS calibration curve and QC samples were prepared for targeted quantitation of MS:MS_COMMENTS tryptophan, serotonin, kynurenine, kynurenic acid, xanthurenic acid, anthranilic MS:MS_COMMENTS acid, indoxyl sulfate, p-cresol sulfate and 3-indole-acetate. 20 µL of each MS:MS_COMMENTS calibrator and QCs were supplemented with 5 µl indoxyl sulfate. Peak areas of MS:MS_COMMENTS each analyte and corresponding internal standard in the calibrator, QCs and MS:MS_COMMENTS samples were integrated using Xcalibur 4.0. A calibration curve was generated by MS:MS_COMMENTS plotting nominal concentration of the analyte in the calibrators versus peak MS:MS_COMMENTS area ratio of analyte #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/ml MS_METABOLITE_DATA_START Samples CKD001 CKD002 CKD003 CKD004 CKD005 CKD006 CKD007 CKD008 Con001 Con002 Con003 Con004 Con005 Con006 Con007 Con008 Factors Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:CKD Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Group:Control Tryptophan 22861 21738 28430 22731 33155 37066 35988 30910 41976 41146 36257 34804 43417 55443 42184 50165 Serotonin 18818 21517 19537 7829 12528 15909 10446 12663 12351 17295 14065 10748 14116 11790 12340 9307 Kynurenine 291 315 308 299 453 553 532 415 298 491 197 182 305 465 430 394 Kynurenic Acid 59 55 36 34 36 73 49 32 42 23 23 15 31 28 42 16 Xanthurenic Acid 16 20 11 16 11 37 24 15 20 8 12 8 17 27 21 11 Anthranilic Acid 6 18 7 5 14 10 10 11 10 10 4 4 6 7 7 13 Indoxyl Sulfate 13956 42623 27598 11213 20435 22474 20020 19183 9721 7478 6720 2954 9203 11268 11949 12961 p-Cresol Sulfate 5684 13118 8270 3710 5136 6569 5845 4850 2335 3048 2762 1115 2248 2336 2562 1654 Indole-3-acetate 114 145 145 72 151 188 151 137 126 82 84 99 234 323 207 188 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pubchem ID Kegg ID Tryptophan 6305 C00078 Serotonin 5202 C00780 Kynurenine 161166 C00328 Kynurenic Acid 3845 C01717 Xanthurenic Acid 5699 C02470 Anthranilic Acid 227 C00108 Indoxyl Sulfate 10258 p-Cresol Sulfate 4615423 Indole-3-acetate 801 C00954 METABOLITES_END #END