#METABOLOMICS WORKBENCH ryant_20200402_063206 DATATRACK_ID:1975 STUDY_ID:ST001353 ANALYSIS_ID:AN002251 PROJECT_ID:PR000923 VERSION 1 CREATED_ON April 3, 2020, 6:25 am #PROJECT PR:PROJECT_TITLE Untargeted metabolomics in skeletal muscle of mice with chronic kidney disease PR:PROJECT_TYPE MS Quantitative analysis PR:PROJECT_SUMMARY This project performed untargeted metabolomics analysis in skeletal muscle PR:PROJECT_SUMMARY (gastrocnemius) in mice with and without chronic kidney disease. PR:INSTITUTE University of Florida PR:DEPARTMENT Applied Physiology 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/NHLBI R01-HL149704 and R01-HL148597 #STUDY ST:STUDY_TITLE Untargeted metabolomics in skeletal muscle of mice with chronic kidney disease ST:STUDY_TYPE MS quantitative analysis ST:STUDY_SUMMARY This study performed untargeted metabolomics analysis of skeletal muscle ST:STUDY_SUMMARY obtained form mice with and without chronic kidney disease. 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 4 ST:TOTAL_SUBJECTS 18 ST:NUM_MALES 8 ST:NUM_FEMALES 10 ST:STUDY_COMMENTS two control male samples processed mistakenly were from soles muscles, while all ST:STUDY_COMMENTS other samples were gastrocnemius muscles. Due to differences in fiber type ST:STUDY_COMMENTS proportions, soleus muscles were not used in final analysis #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 Laboratories 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 gastrocnemius muscle Con009 Group:Control Sex=Male; RAW_FILE_NAME=Con009.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle Con010 Group:Control Sex=Male; RAW_FILE_NAME=Con010.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle Con011 Group:Control Sex=Male; RAW_FILE_NAME=Con011.raw SUBJECT_SAMPLE_FACTORS soleus muscle Con012 Group:Control Sex=Male; RAW_FILE_NAME=Con012.raw SUBJECT_SAMPLE_FACTORS soleus muscle Con013 Group:Control Sex=Male; RAW_FILE_NAME=Con013.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle Con014 Group:Control Sex=Female; RAW_FILE_NAME=Con014.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle Con015 Group:Control Sex=Female; RAW_FILE_NAME=Con015.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle Con016 Group:Control Sex=Female; RAW_FILE_NAME=Con016.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle Con017 Group:Control Sex=Female; RAW_FILE_NAME=Con017.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle Con018 Group:Control Sex=Female; RAW_FILE_NAME=Con018.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD009 Group:CKD Sex=Male; RAW_FILE_NAME=CKD009.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD010 Group:CKD Sex=Male; RAW_FILE_NAME=CKD010.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD011 Group:CKD Sex=Male; RAW_FILE_NAME=CKD011.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD012 Group:CKD Sex=Male; RAW_FILE_NAME=CKD012.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD013 Group:CKD Sex=Male; RAW_FILE_NAME=CKD013.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD014 Group:CKD Sex=Female; RAW_FILE_NAME=CKD014.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD015 Group:CKD Sex=Female; RAW_FILE_NAME=CKD015.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD016 Group:CKD Sex=Female; RAW_FILE_NAME=CKD016.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD017 Group:CKD Sex=Female; RAW_FILE_NAME=CKD017.raw SUBJECT_SAMPLE_FACTORS gastrocnemius muscle CKD018 Group:CKD Sex=Female; RAW_FILE_NAME=CKD018.raw SUBJECT_SAMPLE_FACTORS pooled control male Pooled_Control_Male Group:Control Sex=Male; RAW_FILE_NAME=Pooled_Control_Male.raw SUBJECT_SAMPLE_FACTORS pooled control female Pooled_Control_Female Group:Control Sex=Female; RAW_FILE_NAME=Pooled_Control_Female.raw SUBJECT_SAMPLE_FACTORS pooled CKD male Pooled_CKD_Male Group:CKD Sex=Male; RAW_FILE_NAME=Pooled_CKD_Male.raw SUBJECT_SAMPLE_FACTORS pooled CKD female Pooled_CKD_Female Group:CKD Sex=Female; RAW_FILE_NAME=Pooled_CKD_Female.raw #COLLECTION CO:COLLECTION_SUMMARY Skeletal muscle was quickly dissected, trimmed of fat and connective tissues and CO:COLLECTION_SUMMARY rinsed in PBS to remove and blood. Dissection occurred under ketamine/xylazine CO:COLLECTION_SUMMARY anesthesia and snap frozen in liquid nitrogen. Frozen muscles were stored at CO:COLLECTION_SUMMARY -80C until processing. CO:SAMPLE_TYPE Muscle 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 Muscles were thawed on ice, weighed, and homogenized with a Teflon-tipped SP:SAMPLEPREP_SUMMARY conical pestle with a metal rod (Micro-Tube Sample Pestle with Conical Teflon SP:SAMPLEPREP_SUMMARY Tip, fits 1.5ml Tubes, autoclavable at 121°F; Research Products International SP:SAMPLEPREP_SUMMARY Corp; 199221; Fisher Scientific). The pestle was rinsed with 2-Propanol, water, SP:SAMPLEPREP_SUMMARY and methanol and patted dry with a KimWipe in between samples. The samples were SP:SAMPLEPREP_SUMMARY centrifuged to pellet the tissue debris and protein concentrations were SP:SAMPLEPREP_SUMMARY quantified on the QuBit. The samples were normalized to 500µg/mL of protein SP:SAMPLEPREP_SUMMARY with 5mM Ammonium Acetate in water prior to extraction for a total volume of SP:SAMPLEPREP_SUMMARY 100µL. 25µL of sample was aliquoted into a clean tube and extracted with 5µL SP:SAMPLEPREP_SUMMARY of Global Metabolomics IS and 200µL of 8:1:1 Acetonitrile:Methanol:Acetone to SP:SAMPLEPREP_SUMMARY precipitate proteins. The samples were incubated at 4°C for 30 min and SP:SAMPLEPREP_SUMMARY centrifuged at 20,000xg at 4°C for 10min. 200µL of supernatant was transferred SP:SAMPLEPREP_SUMMARY to a clean Eppendorf tube and dried under nitrogen gas at 30°C and then SP:SAMPLEPREP_SUMMARY reconstituted at 25µL in Global Metabolomics Inj. Std. Mix. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY samples were processed on a Thermo Q-Exactive Oribtrap mass spectrometer with CH:CHROMATOGRAPHY_SUMMARY Dionex UHPLC and autosampler. All samples were analyzed in positive and negative CH:CHROMATOGRAPHY_SUMMARY heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as CH:CHROMATOGRAPHY_SUMMARY separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 CH:CHROMATOGRAPHY_SUMMARY µm column with mobile phase A as 0.1% formic acid in water and mobile phase B CH:CHROMATOGRAPHY_SUMMARY as acetonitrile. This is a polar embedded stationary phase that provides CH:CHROMATOGRAPHY_SUMMARY comprehensive coverage, but does have some limitation is the coverage of very CH:CHROMATOGRAPHY_SUMMARY polar species. The flow rate was 350 µL/min with a column temperature of 25°C. CH:CHROMATOGRAPHY_SUMMARY 4 µL was injected for negative ions and 2 µL for positive ions. 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 CH:SAMPLE_INJECTION 4ul for negative ion, 2ul for positive ion #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 samples were processed on a Thermo Q-Exactive Oribtrap mass spectrometer with MS:MS_COMMENTS Dionex UHPLC and autosampler. All samples were analyzed in positive and negative MS:MS_COMMENTS heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as MS:MS_COMMENTS separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 MS:MS_COMMENTS µm column with mobile phase A as 0.1% formic acid in water and mobile phase B MS:MS_COMMENTS as acetonitrile. This is a polar embedded stationary phase that provides MS:MS_COMMENTS comprehensive coverage, but does have some limitation is the coverage of very MS:MS_COMMENTS polar species. The flow rate was 350 µL/min with a column temperature of 25°C. MS:MS_COMMENTS 4 µL was injected for negative ions and 2 µL for positive ions. Data from MS:MS_COMMENTS positive and negative ion modes were separately subjected to statistical MS:MS_COMMENTS analyses. MZmine (freeware) was used to identify features, deisotope, align MS:MS_COMMENTS features and perform gap filling to fill in any features that may have been MS:MS_COMMENTS missed in the first alignment algorithm. All adducts and complexes were MS:MS_COMMENTS identified and removed from the data set. The primary source of feature MS:MS_COMMENTS identification was performed by mapping against an internal retention time MS:MS_COMMENTS metabolite library established by the SECIM. Additional metabolite searches were MS:MS_COMMENTS performed using HMDB (http://www.hmdb.ca) and the Metabolomics Workbench MS:MS_COMMENTS (https://www.metabolomicsworkbench.org) through a search of the m/z ratio with a MS:MS_COMMENTS [M+H] adduct and a tolerance of 0.002 m/z. MS:MS_RESULTS_FILE ST001353_AN002251_results.txt UNITS:peak height Has m/z:Yes Has RT:No RT units:No RT data #END