{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002398","ANALYSIS_ID":"AN003905","VERSION":"1","CREATED_ON":"December 8, 2022, 4:53 pm"},

"PROJECT":{"PROJECT_TITLE":"Lipidomics of Tango2 Deficient and Wildtype Zebrafish Muscle Tissue","PROJECT_TYPE":"Untargeted LC-MS/MS Lipidomics","PROJECT_SUMMARY":"Rhabdomyolysis is a clinical emergency characterized by severe muscle damage, resulting in the release of intracellular muscle components which leads to myoglobinuria and in severe cases, acute kidney failure. Rhabdomyolysis is caused by genetic factors that are linked to increased disease susceptibility in response to extrinsic triggers. Recessive mutations in TANGO2 result in episodic rhabdomyolysis, metabolic crises, encephalopathy, and cardiac arrhythmia. The underlying mechanism contributing to disease onset in response to specific triggers remains unclear. To address these challenges, we created a zebrafish model of Tango2 deficiency. Here we demonstrate that the loss of Tango2 in zebrafish results in growth defects, early lethality, and increased susceptibility of muscle defects similar to TANGO2 patients. Detailed analyses of skeletal muscle revealed defects in the sarcoplasmic reticulum and mitochondria at the onset of disease development. The sarcoplasmic reticulum (SR) constitutes the primary lipid biosynthesis site and regulates calcium handling in skeletal muscle to control excitation-contraction coupling. Tango2 deficient SR exhibits increased sensitivity to calcium release that was partly restored by inhibition of Ryr1-mediated Ca2+ release in skeletal muscle. Using lipidomics, we identified alterations in the glycerolipid state of tango2 mutants which is critical for membrane stability and energy balance. Therefore, these studies provide insight into key disease processes in Tango2 deficiency and have increased our understanding of the impacts of specific defects on predisposition to environmental triggers in TANGO2-related disorders.","INSTITUTE":"UNC Chapel Hill","DEPARTMENT":"Chemistry","LABORATORY":"MS Core Laboratory","LAST_NAME":"Wallace","FIRST_NAME":"Emily","ADDRESS":"131 South Rd, Chapel Hill, NC, 27514, USA","EMAIL":"emdiane@email.unc.edu","PHONE":"7042453664","FUNDING_SOURCE":"NIH R56AR077017","PUBLICATIONS":"Glycerolipid defects in skeletal muscle contribute to rhabdomyolysis in Tango2 deficiency (submitted to eLife)"},

"STUDY":{"STUDY_TITLE":"Lipidomics of Tango2 Deficient and Wildtype Zebrafish Muscle Tissue","STUDY_TYPE":"Untargeted Lipidomics","STUDY_SUMMARY":"Rhabdomyolysis is a clinical emergency characterized by severe muscle damage, resulting in the release of intracellular muscle components which leads to myoglobinuria and in severe cases, acute kidney failure. Rhabdomyolysis is caused by genetic factors that are linked to increased disease susceptibility in response to extrinsic triggers. Recessive mutations in TANGO2 result in episodic rhabdomyolysis, metabolic crises, encephalopathy, and cardiac arrhythmia. The underlying mechanism contributing to disease onset in response to specific triggers remains unclear. To address these challenges, we created a zebrafish model of Tango2 deficiency. Here we demonstrate that the loss of Tango2 in zebrafish results in growth defects, early lethality, and increased susceptibility of muscle defects similar to TANGO2 patients. Detailed analyses of skeletal muscle revealed defects in the sarcoplasmic reticulum and mitochondria at the onset of disease development. The sarcoplasmic reticulum (SR) constitutes the primary lipid biosynthesis site and regulates calcium handling in skeletal muscle to control excitation-contraction coupling. Tango2 deficient SR exhibits increased sensitivity to calcium release that was partly restored by inhibition of Ryr1-mediated Ca2+ release in skeletal muscle. Using lipidomics, we identified alterations in the glycerolipid state of tango2 mutants which is critical for membrane stability and energy balance. Therefore, these studies provide insight into key disease processes in Tango2 deficiency and have increased our understanding of the impacts of specific defects on predisposition to environmental triggers in TANGO2-related disorders.","INSTITUTE":"University of North Carolina at Chapel Hill","DEPARTMENT":"Chemistry","LABORATORY":"MS Core Laboratory","LAST_NAME":"Wallace","FIRST_NAME":"Emily","ADDRESS":"131 South Rd","EMAIL":"emdiane@email.unc.edu","PHONE":"7042453664","NUM_GROUPS":"2","TOTAL_SUBJECTS":"5","NUM_MALES":"N/A","NUM_FEMALES":"N/A","STUDY_COMMENTS":"Zebrafish were all 4 weeks old when tissue was harvested, sex is determined at 4 weeks old."},

"SUBJECT":{"SUBJECT_TYPE":"Fish","SUBJECT_SPECIES":"Danio rerio","TAXONOMY_ID":"7955","GENOTYPE_STRAIN":"wildtype and tango2bwh211","AGE_OR_AGE_RANGE":"4 weeks"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Z1",
"Factors":{"Genotype":"tango2 Mutant","Weight (mg)":"69.5"},
"Additional sample data":{"RAW_FILE_NAME":"Z1.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z15",
"Factors":{"Genotype":"tango2 Mutant","Weight (mg)":"13.5"},
"Additional sample data":{"RAW_FILE_NAME":"Z15.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z2",
"Factors":{"Genotype":"tango2 Mutant","Weight (mg)":"15"},
"Additional sample data":{"RAW_FILE_NAME":"Z2.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z11",
"Factors":{"Genotype":"tango2 Mutant","Weight (mg)":"8"},
"Additional sample data":{"RAW_FILE_NAME":"Z11.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z22",
"Factors":{"Genotype":"tango2 Mutant","Weight (mg)":"26"},
"Additional sample data":{"RAW_FILE_NAME":"Z22.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z20",
"Factors":{"Genotype":"Wildtype","Weight (mg)":"57.1"},
"Additional sample data":{"RAW_FILE_NAME":"Z20.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z25",
"Factors":{"Genotype":"Wildtype","Weight (mg)":"18.7"},
"Additional sample data":{"RAW_FILE_NAME":"Z25.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z34",
"Factors":{"Genotype":"Wildtype","Weight (mg)":"17"},
"Additional sample data":{"RAW_FILE_NAME":"Z34.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z14",
"Factors":{"Genotype":"Wildtype","Weight (mg)":"9"},
"Additional sample data":{"RAW_FILE_NAME":"Z14.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Z19",
"Factors":{"Genotype":"Wildtype","Weight (mg)":"20"},
"Additional sample data":{"RAW_FILE_NAME":"Z19.mzXML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Fish were bred and maintained using standard methods as described (Westerfield, 2000). All procedures were approved by the Brigham and Women’s Hospital Animal Care and Use Committee. tango2bwh210 and tango2bwh211 zebrafish lines were created in our laboratory by CRISPR-Cas9 approach. Zebrafish embryonic (0-2 days post fertilization) and larval stages (3-45 dpf), juvenile stage (45 dpf-3months) and adults (3 months) have been defined as described previously (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995). All studies presented in this work were performed on tango2bwh211 mutants obtained from heterozygous parents unless specified. Myofibers were isolated from control or tango2 larval zebrafish (45 dpf) as described previously with minor modifications (Ganassi, Zammit, & Hughes, 2021). Skinned zebrafish muscle samples were treated with collagenase for 90 minutes and triturated to release the myofibers. Myofibers were centrifuged at 1000g for 60 sec, washed and resuspended in DMEM media. Myofibers were plated on laminin coated 8 chamber permanox slides (Thermofisher Scientific) for further analysis. Fixed cells were blocked in 10% goat serum/0.3% Triton, incubated in primary antibody overnight at 4ºC, washed in PBS, incubated in secondary antibody for 1 h at room temperature (RT), washed in PBS, then mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Primary antibodies used were anti Tango2 (1:250, 27846-1-AP, Proteintech), mouse monoclonal anti sarcomeric -actinin (1:100, A7732, Millipore Sigma), mouse monoclonal anti Ryr1 (1:100, R129-100UL, Millipore Sigma), anti Tomm20 (1:100, MABT166, Millipore Sigma) and Alexa fluor 568-phalloidin (1:100, Thermo Fisher Scientific, A12380). After washing in PBS several times, samples were incubated with anti-mouse Alexa Fluor (1:100, A-11005) secondary antibody (Thermofisher Scientific). Imaging was performed using a Nikon Ti2 spinning disk confocal microscope.","SAMPLE_TYPE":"Muscle","COLLECTION_METHOD":"Muscle Dissection","STORAGE_CONDITIONS":"Described in summary"},

"TREATMENT":{"TREATMENT_SUMMARY":"Fish were bred and maintained using standard methods as described (Westerfield, 2000). All procedures were approved by the Brigham and Women’s Hospital Animal Care and Use Committee. tango2bwh210 and tango2bwh211 zebrafish lines were created in our laboratory by CRISPR-Cas9 approach. Zebrafish embryonic (0-2 days post fertilization) and larval stages (3-45 dpf), juvenile stage (45 dpf-3months) and adults (3 months) have been defined as described previously (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995). All studies presented in this work were performed on tango2bwh211 mutants obtained from heterozygous parents unless specified."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Control and tango2 mutant myofibers (4 weeks, n=5 each) were homogenized with 1 ml of MBTE. 300 µL of methanol with internal standard was added and samples were mixed for 10 minutes. 200 µL of water was added to facilitate phase separation. The extracts were centrifuged at 20,000 rcf for 10 minutes. The top layer was removed, dried and reconstituted in 150 µl of IPA for analysis.","PROCESSING_METHOD":"Liquid/Liquid Partition","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACTION_METHOD":"Liquid/Liquid Partition","EXTRACT_CLEANUP":"Centrifugation (20,000 rcf for 10 min)","EXTRACT_STORAGE":"-80℃","SAMPLE_RESUSPENSION":"150 µL of IPA","SAMPLE_DERIVATIZATION":"none","SAMPLE_SPIKING":"Spiked with 1.5 µg/mL of deuterated internal standards (Avanti EquiSplash mix)"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Analysis was performed using a Thermo Q Exactive Plus coupled to a Waters Acquity H-Class LC. A 100 mm x 2.1 mm, 2.1 µm Waters BEH C18 column was used for separations. The following mobile phases were used: A- 60/40 ACN/H20 B- 90/10 IPA/ACN; both mobile phases contained 10 mM Ammonium Formate and 0.1% Formic Acid. A flow rate of 0.2 mL/minutes was used. Starting composition was 32% B, which increased to 40% B at 1 minute (held until 1.5 minutes) then 45% B at 4 minutes. This was increased to 50% B at 5 minutes, 60% B at 8 minutes, 70% B at 11 minutes, and 80% B at 14 minutes (held until 16 minutes). At 16 minutes the composition switched back to starting conditions (32% B) and was held for 4 minutes to re-equilibrate the column. Samples were analyzed in positive/negative switching ionization mode with top 5 data dependent fragmentation.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity H-Class","COLUMN_NAME":"Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)","SOLVENT_A":"60/40 ACN/H20 10 mM ammonium formate","SOLVENT_B":"90/10 IPA/ACN 10 mM ammonium formate","FLOW_GRADIENT":"Starting composition was 32% B, which increased to 40% B at 1 minute (held until 1.5 minutes) then 45% B at 4 minutes. This was increased to 50% B at 5 minutes, 60% B at 8 minutes, 70% B at 11 minutes, and 80% B at 14 minutes (held until 16 minutes). At 16 minutes the composition switched back to starting conditions (32% B) and was held for 4 minutes to re-equilibrate the column.","FLOW_RATE":"0.2 mL/min","COLUMN_TEMPERATURE":"45","COLUMN_PRESSURE":"4220 psi","INJECTION_TEMPERATURE":"10","INTERNAL_STANDARD":"Avanti EquiSplash mix","SAMPLE_INJECTION":"5 µL","CAPILLARY_VOLTAGE":"3.50 KV","RUNNING_VOLTAGE":"3.50 KV","WEAK_WASH_SOLVENT_NAME":"90:10 Water:Acetontrile","WEAK_WASH_VOLUME":"300 µL","STRONG_WASH_SOLVENT_NAME":"1:1 Methanol:Isopropanol","STRONG_WASH_VOLUME":"50 µL","SAMPLE_LOOP_SIZE":"10 µL","SAMPLE_SYRINGE_SIZE":"100 µL"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"UNC MS Core Laboratory","OPERATOR_NAME":"Emily Wallace","DETECTOR_TYPE":"Orbitrap","SOFTWARE_VERSION":"LipidSearch 4.2","ACQUISITION_DATE":"11/02/2022","DATA_FORMAT":".Raw"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF-X Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Analysis was performed using a Thermo Q Exactive Plus coupled to a Waters Acquity H-Class LC. A 100 mm x 2.1 mm, 2.1 µm Waters BEH C18 column was used for separations. The following mobile phases were used: A- 60/40 ACN/H20 B- 90/10 IPA/ACN; both mobile phases contained 10 mM Ammonium Formate and 0.1% Formic Acid. A flow rate of 0.2 mL/minutes was used. Starting composition was 32% B, which increased to 40% B at 1 minute (held until 1.5 minutes) then 45% B at 4 minutes. This was increased to 50% B at 5 minutes, 60% B at 8 minutes, 70% B at 11 minutes, and 80% B at 14 minutes (held until 16 minutes). At 16 minutes the composition switched back to starting conditions (32% B) and was held for 4 minutes to re-equilibrate the column. Samples were analyzed in positive/negative switching ionization mode with top 5 data dependent fragmentation. Raw data were analyzed by LipidSearch 4.2. Lipids were identified by MS2 fragmentation (mass error of precursor=5 ppm, mass error of product=8 ppm). The identifications were generated individually for each sample and then aligned by grouping the samples (OxPAPC=C, HF=S1, Con=S2). Normalization was performed using EquiSplash from Avanti. Samples were normalized and biological replicates were averaged. P-value and fold change were calculated as instructed as previously described (Aguilan, Kulej, & Sidoli, 2020). P-value was set to 0.05.","CAPILLARY_TEMPERATURE":"300","CAPILLARY_VOLTAGE":"3.50 KV","COLLISION_ENERGY":"25, 35, 45 V stepped collision energy","FRAGMENTATION_METHOD":"HCD","ION_SOURCE_TEMPERATURE":"400","ION_SPRAY_VOLTAGE":"3.50 KV","IONIZATION":"HESI","MASS_ACCURACY":"5 ppm","REAGENT_GAS":"Nitrogen","SOURCE_TEMPERATURE":"400","SPRAY_VOLTAGE":"3.5 KV"},

"MS_METABOLITE_DATA":{
"Units":"peak area",

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e":"TG(47:13)+NH4_1"},{"metabolite_name":"TG(48:1)+H"},{"metabolite_name":"TG(51:7)+NH4"},{"metabolite_name":"TG(52:1)+H"},{"metabolite_name":"TG(52:5)+Na"},{"metabolite_name":"TG(53:10)+NH4"},{"metabolite_name":"TG(54:1)+H"},{"metabolite_name":"TG(54:11)+NH4"},{"metabolite_name":"TG(54:12)+NH4"},{"metabolite_name":"TG(55:11)+NH4"},{"metabolite_name":"TG(60:11)+NH4"},{"metabolite_name":"TG(62:11)+NH4"},{"metabolite_name":"TG(8:0_12:0_14:0)+NH4"},{"metabolite_name":"WE(4:0_18:3)+H"},{"metabolite_name":"ZyE(16:0)+NH4"},{"metabolite_name":"ZyE(32:0)+H"},{"metabolite_name":"ZyE(34:0)+H"}]
}

}