#METABOLOMICS WORKBENCH dongf01_20200326_193205 DATATRACK_ID:1958 STUDY_ID:ST001338 ANALYSIS_ID:AN002232 PROJECT_ID:000000 VERSION 1 CREATED_ON April 3, 2020, 9:39 am #PROJECT PR:PROJECT_TITLE aryl hydrocarbon receptor-related compounds studies PR:PROJECT_SUMMARY The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor PR:PROJECT_SUMMARY that responds to a variety of structurally diverse exogenous and endogenous PR:PROJECT_SUMMARY small molecules. Gut microbiota utilizing tryptophan and indole metabolism as a PR:PROJECT_SUMMARY reservoir, has been demonstrated to provide an abundant source of AHR ligands. PR:PROJECT_SUMMARY So differential analysis was performed to find the potential candidates of AHR PR:PROJECT_SUMMARY activator in cecal contents between conventional and germ-free mice with the PR:PROJECT_SUMMARY help of untargeted global profiling. PR:INSTITUTE Pennsylvania State University PR:LAST_NAME DONG PR:FIRST_NAME FANGCONG PR:ADDRESS 314 Life Sciences Building, University Park, PA 16802 PR:EMAIL fxd93@psu.edu PR:PHONE 8148637610 #STUDY ST:STUDY_TITLE Global profiling for cecal contents ST:STUDY_SUMMARY The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor ST:STUDY_SUMMARY that responds to a variety of structurally diverse exogenous and endogenous ST:STUDY_SUMMARY small molecules. Gut microbiota utilizing tryptophan and indole metabolism as a ST:STUDY_SUMMARY reservoir, has been demonstrated to provide an abundant source of AHR ligands. ST:STUDY_SUMMARY So differential analysis was performed to find the potential candidates of AHR ST:STUDY_SUMMARY activator in cecal contents between conventional and germ-free mice with the ST:STUDY_SUMMARY help of untargeted global profiling. ST:INSTITUTE Pennsylvania State University ST:LAST_NAME DONG ST:FIRST_NAME FANGCONG ST:ADDRESS 314 Life Sciences Building, University Park, PA 16802 ST:EMAIL fxd93@psu.edu ST:PHONE 8148637610 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - Cecal content_C1 Genotype:wild-type Treatment=chow diet; RAW_FILE_NAME=Cecal content_C1.raw SUBJECT_SAMPLE_FACTORS - Cecal content_C2 Genotype:wild-type Treatment=chow diet; RAW_FILE_NAME=Cecal content_C2.raw SUBJECT_SAMPLE_FACTORS - Cecal content_C3 Genotype:wild-type Treatment=chow diet; RAW_FILE_NAME=Cecal content_C3.raw SUBJECT_SAMPLE_FACTORS - Cecal content_C4 Genotype:wild-type Treatment=chow diet; RAW_FILE_NAME=Cecal content_C4.raw SUBJECT_SAMPLE_FACTORS - Cecal content_GF1 Genotype:germ-free Treatment=chow diet; RAW_FILE_NAME=Cecal content_GF1.raw SUBJECT_SAMPLE_FACTORS - Cecal content_GF2 Genotype:germ-free Treatment=chow diet; RAW_FILE_NAME=Cecal content_GF2.raw SUBJECT_SAMPLE_FACTORS - Cecal content_GF3 Genotype:germ-free Treatment=chow diet; RAW_FILE_NAME=Cecal content_GF3.raw SUBJECT_SAMPLE_FACTORS - Cecal content_GF4 Genotype:germ-free Treatment=chow diet; RAW_FILE_NAME=Cecal content_GF4.raw #COLLECTION CO:COLLECTION_SUMMARY C57BL/6J wild type mice were originally purchased from Jackson Laboratories (Bar CO:COLLECTION_SUMMARY Harbor, MN, USA). Germ-free (GF) C57BL/6J mice were from the Pennsylvania State CO:COLLECTION_SUMMARY University Gnotobiotic Animal Research Facility. Mice were bred in-house and fed CO:COLLECTION_SUMMARY on a standard animal chow diet. Animal experiments were performed after approval CO:COLLECTION_SUMMARY by the Institutional Animal Care and Use Committee. Fresh cecal contents from CO:COLLECTION_SUMMARY conventional and GF mice were collected and stored at -80 °C. CO:SAMPLE_TYPE Cecum #TREATMENT TR:TREATMENT_SUMMARY C57BL/6J wild type mice were originally purchased from Jackson Laboratories (Bar TR:TREATMENT_SUMMARY Harbor, MN, USA). Germ-free (GF) C57BL/6J mice were from the Pennsylvania State TR:TREATMENT_SUMMARY University Gnotobiotic Animal Research Facility. Mice were bred in-house and fed TR:TREATMENT_SUMMARY on a standard animal chow diet. Animal experiments were performed after approval TR:TREATMENT_SUMMARY by the Institutional Animal Care and Use Committee. Fresh cecal contents from TR:TREATMENT_SUMMARY conventional and GF mice were collected and stored at -80 °C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Each mixture was homogenized with 1 mm zirconium beads using a BeadBlasterTM 24 SP:SAMPLEPREP_SUMMARY (Benchmark Scientific, Edison, NJ, USA) homogenizer. All samples were SP:SAMPLEPREP_SUMMARY homogenized according to the program parameters: 6500 - 1×30 - 005 (×3). After SP:SAMPLEPREP_SUMMARY vortexing, samples were sonicated for 20 min in an ice water bath, prior to SP:SAMPLEPREP_SUMMARY centrifugation at 20,000 × g for 20 min at 4 ℃. The supernatants were SP:SAMPLEPREP_SUMMARY collected, dried in a Savant SpeedVac (Thermo Scientific, Waltham, MA, USA), and SP:SAMPLEPREP_SUMMARY reconstituted in 100 μL of 3% methanol (v/v) containing 1 µM chlorpropamide SP:SAMPLEPREP_SUMMARY (internal standard). SP:SAMPLEPREP_PROTOCOL_FILENAME MS_protocol_for_global_profiling.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters Acquity BEH C18 (100 x 2mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Tribrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Solvent A was HPLC-grade water with 0.1% formic acid, and solvent B was MS:MS_COMMENTS HPLC-grade acetonitrile with 0.1% formic acid. The initial condition was 97% A MS:MS_COMMENTS and 3% B, increasing to 45% B at 10 min and 75% B at 12 min, where it was held MS:MS_COMMENTS at 75% B until 17.5 min before returning to the initial conditions. Differential MS:MS_COMMENTS analyses were performed by the use of Compound Discoverer (Thermo Fisher MS:MS_COMMENTS Scientific, Waltham, MA, USA) MS:MS_RESULTS_FILE ST001338_AN002232_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END