{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001353","ANALYSIS_ID":"AN002251","VERSION":"1","CREATED_ON":"April 3, 2020, 6:25 am"},

"PROJECT":{"PROJECT_TITLE":"Untargeted metabolomics in skeletal muscle of mice with chronic kidney disease","PROJECT_TYPE":"MS Quantitative analysis","PROJECT_SUMMARY":"This project performed untargeted metabolomics analysis in skeletal muscle (gastrocnemius) in mice with and without chronic kidney disease.","INSTITUTE":"University of Florida","DEPARTMENT":"Applied Physiology and Kinesiology","LAST_NAME":"Ryan","FIRST_NAME":"Terence","ADDRESS":"1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA","EMAIL":"ryant@ufl.edu","PHONE":"352-294-1700","FUNDING_SOURCE":"NIH/NHLBI R01-HL149704 and R01-HL148597"},

"STUDY":{"STUDY_TITLE":"Untargeted metabolomics in skeletal muscle of mice with chronic kidney disease","STUDY_TYPE":"MS quantitative analysis","STUDY_SUMMARY":"This study performed untargeted metabolomics analysis of skeletal muscle obtained form mice with and without chronic kidney disease.","INSTITUTE":"University of Florida","LAST_NAME":"Ryan","FIRST_NAME":"Terence","ADDRESS":"1864 Stadium Rd, FLG 114, Gainesville, FL, 32611, USA","EMAIL":"ryant@ufl.edu","PHONE":"352-294-1700","NUM_GROUPS":"4","TOTAL_SUBJECTS":"18","NUM_MALES":"8","NUM_FEMALES":"10","STUDY_COMMENTS":"two control male samples processed mistakenly were from soles muscles, while all other samples were gastrocnemius muscles. Due to differences in fiber type proportions, soleus muscles were not used in final analysis"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"C57BL6J","AGE_OR_AGE_RANGE":"18-20 weeks","WEIGHT_OR_WEIGHT_RANGE":"20-30g","GENDER":"Male and female","ANIMAL_ANIMAL_SUPPLIER":"Jackson Laboratories","ANIMAL_HOUSING":"5/cage","ANIMAL_LIGHT_CYCLE":"12h","ANIMAL_FEED":"Ad libitum. Control mice received custom casein-diet. Chronic kidney disease was induced by supplementing casein-based diet with 0.15% adenine","ANIMAL_WATER":"Ad libitum"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"Con009",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"Con009.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"Con010",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"Con010.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"Con011",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"Con011.raw"}
},
{
"Subject ID":"soleus muscle",
"Sample ID":"Con012",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"Con012.raw"}
},
{
"Subject ID":"soleus muscle",
"Sample ID":"Con013",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"Con013.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"Con014",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"Con014.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"Con015",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"Con015.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"Con016",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"Con016.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"Con017",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"Con017.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"Con018",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"Con018.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD009",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"CKD009.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD010",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"CKD010.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD011",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"CKD011.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD012",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"CKD012.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD013",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"CKD013.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD014",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"CKD014.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD015",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"CKD015.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD016",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"CKD016.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD017",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"CKD017.raw"}
},
{
"Subject ID":"gastrocnemius muscle",
"Sample ID":"CKD018",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"CKD018.raw"}
},
{
"Subject ID":"pooled control male",
"Sample ID":"Pooled_Control_Male",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"Pooled_Control_Male.raw"}
},
{
"Subject ID":"pooled control female",
"Sample ID":"Pooled_Control_Female",
"Factors":{"Group":"Control"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"Pooled_Control_Female.raw"}
},
{
"Subject ID":"pooled CKD male",
"Sample ID":"Pooled_CKD_Male",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Male","RAW_FILE_NAME":"Pooled_CKD_Male.raw"}
},
{
"Subject ID":"pooled CKD female",
"Sample ID":"Pooled_CKD_Female",
"Factors":{"Group":"CKD"},
"Additional sample data":{"Sex":"Female","RAW_FILE_NAME":"Pooled_CKD_Female.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Skeletal muscle was quickly dissected, trimmed of fat and connective tissues and rinsed in PBS to remove and blood. Dissection occurred under ketamine/xylazine anesthesia and snap frozen in liquid nitrogen. Frozen muscles were stored at -80C until processing.","SAMPLE_TYPE":"Muscle","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"We utilized an established adenine-diet model to induce CKD in mice. Mice were assigned to a casein-based chow diet for 7 days, followed by induction of renal tubular injury by supplementing the diet with 0.2% adenine for 7 days, and were subsequently maintained on a 0.15% adenine diet for 7 more weeks. CKD mice were then placed back on control casein diet for 2 weeks to prior to euthanasia and terminal experiments. Control mice received casein diet for the duration of the study.","ANIMAL_ANESTHESIA":"Ketamine/Xylazine","ANIMAL_ENDP_EUTHANASIA":"Ketamine/Xylazine"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Muscles were thawed on ice, weighed, and homogenized with a Teflon-tipped conical pestle with a metal rod (Micro-Tube Sample Pestle with Conical Teflon Tip, fits 1.5ml Tubes, autoclavable at 121°F; Research Products International Corp; 199221; Fisher Scientific). The pestle was rinsed with 2-Propanol, water, and methanol and patted dry with a KimWipe in between samples. The samples were centrifuged to pellet the tissue debris and protein concentrations were quantified on the QuBit. The samples were normalized to 500µg/mL of protein with 5mM Ammonium Acetate in water prior to extraction for a total volume of 100µL. 25µL of sample was aliquoted into a clean tube and extracted with 5µL of Global Metabolomics IS and 200µL of 8:1:1 Acetonitrile:Methanol:Acetone to precipitate proteins. The samples were incubated at 4°C for 30 min and centrifuged at 20,000xg at 4°C for 10min. 200µL of supernatant was transferred to a clean Eppendorf tube and dried under nitrogen gas at 30°C and then reconstituted at 25µL in Global Metabolomics Inj. Std. Mix."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"samples were processed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 µm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is a polar embedded stationary phase that provides comprehensive coverage, but does have some limitation is the coverage of very polar species. The flow rate was 350 µL/min with a column temperature of 25°C. 4 µL was injected for negative ions and 2 µL for positive ions.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex","COLUMN_NAME":"ACE 5 C18-300 (100 x 2.1mm)","FLOW_RATE":"350ul/min","COLUMN_TEMPERATURE":"25C","SOLVENT_A":"0.1% formic acid","SOLVENT_B":"acetonitrile","SAMPLE_INJECTION":"4ul for negative ion, 2ul for positive ion"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"samples were processed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 µm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is a polar embedded stationary phase that provides comprehensive coverage, but does have some limitation is the coverage of very polar species. The flow rate was 350 µL/min with a column temperature of 25°C. 4 µL was injected for negative ions and 2 µL for positive ions. Data from positive and negative ion modes were separately subjected to statistical analyses. MZmine (freeware) was used to identify features, deisotope, align features and perform gap filling to fill in any features that may have been missed in the first alignment algorithm. All adducts and complexes were identified and removed from the data set. The primary source of feature identification was performed by mapping against an internal retention time metabolite library established by the SECIM. Additional metabolite searches were performed using HMDB (http://www.hmdb.ca) and the Metabolomics Workbench (https://www.metabolomicsworkbench.org) through a search of the m/z ratio with a [M+H] adduct and a tolerance of 0.002 m/z.","MS_RESULTS_FILE":"ST001353_AN002251_results.txt UNITS:peak height Has m/z:Yes Has RT:No RT units:No RT data"}

}