{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002706","ANALYSIS_ID":"AN004388","VERSION":"1","CREATED_ON":"May 18, 2023, 11:24 am"},

"PROJECT":{"PROJECT_TITLE":"Changes in the plasma metabolomics en in the muscle-specific rescue of Bmal1","PROJECT_SUMMARY":"This study examines the systemic effects of muscle-specific Bmal1 expression in the Bmal1-KO mouse model. We used an adeno-associated virus to rescue the expression of the clock gene Bmal1 in skeletal muscle of the Bmal1 KO.","INSTITUTE":"University of Florida","DEPARTMENT":"Department of Physiology and Aging","LAST_NAME":"Esser","FIRST_NAME":"Karyn","ADDRESS":"1345 Center Drive, M552, Gainesville, Florida, 32610, USA","EMAIL":"kaesser@ufl.edu","PHONE":"352-273-5728"},

"STUDY":{"STUDY_TITLE":"Plasma metabolomics of Bmal1-KO and Bmal1-KO+AAV","STUDY_SUMMARY":"Disruption of the circadian clock in skeletal muscle worsens local and systemic health, leading to decreased muscle strength, metabolic dysfunction, and aging-like phenotypes. Whole-body knockout mice that lack Bmal1, a key component of the molecular clock, display premature aging. Here, by using adeno-associated viruses, we rescued Bmal1 expression specifically in the skeletal muscle of Bmal1-KO mice and found that this improves their healthspan and lifespan. Plasma samples from 40-week-old KO and KO+AAV male mice were collected at ZT1 to characterize the systemic effects of muscle-specific expression of Bmal1. Overall, our findings highlight the critical role of skeletal muscle in systemic health.","INSTITUTE":"University of Florida","LAST_NAME":"Esser","FIRST_NAME":"Karyn","ADDRESS":"1345 Center Drive, M552, Gainesville, Florida, 32610, USA","EMAIL":"kaesser@ufl.edu","PHONE":"352-273-5728"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"B6.129-Arntltm1Bra/J","AGE_OR_AGE_RANGE":"40 weeks","GENDER":"Male","ANIMAL_FEED":"Ad libitum","ANIMAL_WATER":"Ad libitum"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"26",
"Sample ID":"PL5",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_29[PL5]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_29[PL5]n.mzXML"}
},
{
"Subject ID":"238",
"Sample ID":"PL6",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_30[PL6]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_30[PL6]n.mzXML"}
},
{
"Subject ID":"205",
"Sample ID":"PL7",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_31[PL7]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_31[PL7]n.mzXML"}
},
{
"Subject ID":"247",
"Sample ID":"PL8",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_32[PL8]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_32[PL8]n.mzXML"}
},
{
"Subject ID":"82",
"Sample ID":"PL9",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"AAV"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_33[PL9]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_33[PL9]n.mzXML"}
},
{
"Subject ID":"143",
"Sample ID":"PL10",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"AAV"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_34[PL10]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_34[PL10]n.mzXML"}
},
{
"Subject ID":"144",
"Sample ID":"PL11",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"AAV"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_35[PL11]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_35[PL11]n.mzXML"}
},
{
"Subject ID":"146",
"Sample ID":"PL12",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"AAV"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_36[PL12]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_36[PL12]n.mzXML"}
},
{
"Subject ID":"Pool1",
"Sample ID":"Pool1",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_1[Pool_8]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_1[Pool_8]n.mzXML"}
},
{
"Subject ID":"Pool2",
"Sample ID":"Pool2",
"Factors":{"Genotype":"Bmal1-KO","Treatment":"AAV"},
"Additional sample data":{"RAW_FILE_NAME":"QE2_jdg_341_Esser_1[Pool_9]p.mzXML  RAW_FILE_NAME":"QE2_jdg_341_Esser_1[Pool_9]n.mzXML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Mice were anesthetized using isofluorane. Blood was collected by intracardiac puncture. EDTA was used as anticoagulant. Blood was centrifuged at 2,000xg for 15 mins and plasma was collected and subsequently frozen in liquid nitrogen. Samples are stored at -80°C.","SAMPLE_TYPE":"Blood (plasma)","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Bmal1-KO+AAV mice were systemically infected in the subxiphoid region with 20μl containing 2 × 10^11 genome copies of AAV9 on postnatal day 5."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"All provided samples were extracted following our cellular extraction procedure with pre-normalization to the sample protein content. Each extract was spiked with 1 ul of standard mixture consisting of deuterium labeled carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, myristoylcarnitine and palmitoylcarnitine to targeted identification of carnitines in the sample. Metabolite extraction was done by protein precipitation using 200 µl of 8:1:1 Acetonitrile: Methanol: Acetone with 0.1% formic acid. Further protein precipitation was allowed by incubating the samples at 4°C for 20 min. Samples were placed in an ultrasonic bath for 10 min and then centrifuged at 20 000 xg for 5min at 4°C to pellet the protein. 190 µl supernatant was transferred from each sample into clean tube and dried under a gentle stream of nitrogen at 30°C. The dried extracts were re-suspended with 25 µL water with 0.1% formic acid. Resuspension was allowed at 4°C for 10 -15 min then samples were centrifuged at 20000 xg for 5 min at 4°C. Supernatants were transferred into clean LC-vials for targeted LC-MS quantitation on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Global metabolomics profiling was performed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. Each extract was spiked with 1 ul of standard mixture consisting of deuterium labeled carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, myristoylcarnitine and palmitoylcarnitine to targeted identification of carnitines in the sample. 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 2 Excel-C18 pfp, 100x2.1mm, 2um 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)","SOLVENT_A":"100% water; 0.1% formic acid","SOLVENT_B":"100% acetonitrile","SOLVENT_C":"-","FLOW_GRADIENT":"-","FLOW_RATE":"350 µL/min","COLUMN_TEMPERATURE":"25°C"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"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.","MS_RESULTS_FILE":"ST002706_AN004388_Results.txt UNITS:Peak height Has m/z:Yes Has RT:Yes RT units:Minutes"}

}