#METABOLOMICS WORKBENCH chamberlainca_20190830_095832 DATATRACK_ID:1809 STUDY_ID:ST001263 ANALYSIS_ID:AN002099 PROJECT_ID:PR000848 VERSION 1 CREATED_ON October 8, 2019, 1:27 pm #PROJECT PR:PROJECT_TITLE Metabolomic Profiling of Oxalate-Degrading Probiotic Lactobacillus acidophilus PR:PROJECT_TITLE and Lactobacillus gasseri PR:PROJECT_TYPE Metabolomic/Lipidomic Profiling PR:PROJECT_SUMMARY Metabolomic and lipidomic profiling of Lactobacillus acidophilus and PR:PROJECT_SUMMARY Lactobacillus gasseri PR:INSTITUTE University of Florida PR:DEPARTMENT Pathology, Immunology and Laboratory Medicine PR:LABORATORY Timothy J. Garrett, PhD PR:LAST_NAME Chamberlain PR:FIRST_NAME Casey PR:ADDRESS UF Dept of Pathology PO Box 100275 Gainesville, FL 32610 PR:EMAIL chamberlainc@ufl.edu PR:PHONE 3522735720 PR:FUNDING_SOURCE NIH #STUDY ST:STUDY_TITLE Metabolomic Profiling of Oxalate-Degrading Probiotic Lactobacillus acidophilus ST:STUDY_TITLE and Lactobacillus gasseri ST:STUDY_TYPE Metabolomic/Lipidomic Profiling ST:STUDY_SUMMARY Metabolomic and lipidomic profiling of Lactobacillus acidophilus and ST:STUDY_SUMMARY Lactobacillus gasseri to identify unique differences in their biochemistry that ST:STUDY_SUMMARY could potentially influence their ability to serve as probiotic agents for ST:STUDY_SUMMARY oxalate diseases. ST:INSTITUTE University of Florida ST:DEPARTMENT Pathology, Immunology and Laboratory Medicine ST:LABORATORY Timothy J. Garrett, PhD ST:LAST_NAME Chamberlain ST:FIRST_NAME Casey ST:ADDRESS UF Dept of Pathology PO Box 100275 Gainesville, FL 32610 ST:EMAIL chamberlainc@ufl.edu ST:PHONE 3522735720 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 16 ST:NUM_MALES N/A ST:NUM_FEMALES N/A #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Lactobacillus SU:TAXONOMY_ID 1578 SU:GENDER Not applicable SU:CELL_BIOSOURCE_OR_SUPPLIER American Type Culture Collection (ATCC) SU:CELL_STRAIN_DETAILS L. acidophilus (ATCC 4357), L. gasseri (ATCC 33323) #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - L. acidophilus_1 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. acidophilus_2 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. acidophilus_3 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. acidophilus_4 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. acidophilus_5 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. acidophilus_6 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. acidophilus_7 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. acidophilus_8 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. gasseri_1 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. gasseri_2 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. gasseri_3 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. gasseri_4 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. gasseri_5 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. gasseri_6 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. gasseri_7 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics SUBJECT_SAMPLE_FACTORS - L. gasseri_8 Factor:data1 Concentration (mg/mL)=75; Analyses=Metabolomics, Lipidomics #COLLECTION CO:COLLECTION_SUMMARY Cultured in MRS media from frozen glycerol stocks for 24 hours. Harvested, CO:COLLECTION_SUMMARY washed, and resuspended in PBS-based buffer solution. Individual cultures CO:COLLECTION_SUMMARY treated as biological replicates. CO:SAMPLE_TYPE Bacterial cells CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_TUBE_TEMP -80C #TREATMENT TR:TREATMENT_SUMMARY No treatment. Simply a metabolomic comparison between species. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Normalized to 75 mg/mL in PBS-based buffer solution. Metabolites extracted by SP:SAMPLEPREP_SUMMARY protein precipitation using 8:1:1 acetonitrile:methanol:acetone. Lipids SP:SAMPLEPREP_SUMMARY extracted using the Folch method with 4:2:1 chloroform:methanol:water. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Gradient elution was performed with 60:40 acetonitrile:water with 0.1% formic CH:CHROMATOGRAPHY_SUMMARY acid and 10mM ammonium formate as solvent A and 90:8:2 CH:CHROMATOGRAPHY_SUMMARY isopropanol:acetonitrile:water with 0.1% formic acid and 10mM ammonium formate CH:CHROMATOGRAPHY_SUMMARY as solvent B, at a flow rate of 0.5 mL/min, as such: 0 - 1 min: 80% A, 1 - 3 CH:CHROMATOGRAPHY_SUMMARY min: 80% → 70% A, 3 - 4 min: 70% → 55% A, 4 - 6 min: 55% → 40% A, 6 - 8 CH:CHROMATOGRAPHY_SUMMARY min: 40% → 35% A, 8 - 10 min: 35% A, 10 -15 min: 35% → 10% A, 15 - 17 min: CH:CHROMATOGRAPHY_SUMMARY 10% → 2% A, 17 - 18 min: 2% A, 18 - 19 min: 2% → 80% A, 19 - 23 min: 80% A CH:CHROMATOGRAPHY_SUMMARY (column flush & equilibration) CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 50 CH:SOLVENT_A 60:40 acetonitrile:water + 0.1% formic acid + 10mM ammonium formate CH:SOLVENT_B 90:8:2 isopropanol:acetonitrile:water + 0.1% formic acid + 10mM ammonium formate #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS See manuscript MS:MS_RESULTS_FILE ST001263_AN002099_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END