{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001887","ANALYSIS_ID":"AN003055","VERSION":"1","CREATED_ON":"July 30, 2021, 1:00 pm"},

"PROJECT":{"PROJECT_TITLE":"Multi-omics study of hypertrophic cardiomyopathy","PROJECT_SUMMARY":"Multi-omics study of human heart tissues in the context of hypertrophic cardiomyopathy","INSTITUTE":"Stanford University","LAST_NAME":"Contrepois","FIRST_NAME":"Kevin","ADDRESS":"300 Pasteur Dr","EMAIL":"kcontrep@stanford.edu","PHONE":"6506664538"},

"STUDY":{"STUDY_TITLE":"Untargeted lipidomics of hypertrophic cardiomyopathy (part II)","STUDY_SUMMARY":"Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure-function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. In this work, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM.","INSTITUTE":"Stanford University","LAST_NAME":"Contrepois","FIRST_NAME":"Kevin","ADDRESS":"300 Pasteur Dr","EMAIL":"kcontrep@stanford.edu","PHONE":"6506664538"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"D1234",
"Factors":{"Group":"Donor"},
"Additional sample data":{"SampleID":"D1","RAW_FILE_NAME":"pRPLC_D1234","RAW_FILE_NAME":"nRPLC_D1234"}
},
{
"Subject ID":"-",
"Sample ID":"D1331",
"Factors":{"Group":"Donor"},
"Additional sample data":{"SampleID":"D3","RAW_FILE_NAME":"pRPLC_D1331","RAW_FILE_NAME":"nRPLC_D1331"}
},
{
"Subject ID":"-",
"Sample ID":"D2507",
"Factors":{"Group":"Donor"},
"Additional sample data":{"SampleID":"D8","RAW_FILE_NAME":"pRPLC_D2507","RAW_FILE_NAME":"nRPLC_D2507"}
},
{
"Subject ID":"-",
"Sample ID":"D2540",
"Factors":{"Group":"Donor"},
"Additional sample data":{"SampleID":"D9","RAW_FILE_NAME":"pRPLC_D2540","RAW_FILE_NAME":"nRPLC_D2540"}
},
{
"Subject ID":"-",
"Sample ID":"D2552",
"Factors":{"Group":"Donor"},
"Additional sample data":{"SampleID":"D10","RAW_FILE_NAME":"pRPLC_D2552","RAW_FILE_NAME":"nRPLC_D2552"}
},
{
"Subject ID":"-",
"Sample ID":"D2554",
"Factors":{"Group":"Donor"},
"Additional sample data":{"SampleID":"D11","RAW_FILE_NAME":"pRPLC_D2554","RAW_FILE_NAME":"nRPLC_D2554"}
},
{
"Subject ID":"-",
"Sample ID":"M433",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H4","RAW_FILE_NAME":"pRPLC_M433","RAW_FILE_NAME":"nRPLC_M433"}
},
{
"Subject ID":"-",
"Sample ID":"M467",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H5","RAW_FILE_NAME":"pRPLC_M467","RAW_FILE_NAME":"nRPLC_M467"}
},
{
"Subject ID":"-",
"Sample ID":"M1385",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H6","RAW_FILE_NAME":"pRPLC_M1385","RAW_FILE_NAME":"nRPLC_M1385"}
},
{
"Subject ID":"-",
"Sample ID":"M1455",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H7","RAW_FILE_NAME":"pRPLC_M1455","RAW_FILE_NAME":"nRPLC_M1455"}
},
{
"Subject ID":"-",
"Sample ID":"M2622",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H8","RAW_FILE_NAME":"pRPLC_M2622","RAW_FILE_NAME":"nRPLC_M2622"}
},
{
"Subject ID":"-",
"Sample ID":"M2673",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H11","RAW_FILE_NAME":"pRPLC_M2673","RAW_FILE_NAME":"nRPLC_M2673"}
},
{
"Subject ID":"-",
"Sample ID":"M2692",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H13","RAW_FILE_NAME":"pRPLC_M2692","RAW_FILE_NAME":"nRPLC_M2692"}
},
{
"Subject ID":"-",
"Sample ID":"M2799",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H16","RAW_FILE_NAME":"pRPLC_M2799","RAW_FILE_NAME":"nRPLC_M2799"}
},
{
"Subject ID":"-",
"Sample ID":"M2800",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H17","RAW_FILE_NAME":"pRPLC_M2800","RAW_FILE_NAME":"nRPLC_M2800"}
},
{
"Subject ID":"-",
"Sample ID":"M2803",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H18","RAW_FILE_NAME":"pRPLC_M2803","RAW_FILE_NAME":"nRPLC_M2803"}
},
{
"Subject ID":"-",
"Sample ID":"M2856",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H21","RAW_FILE_NAME":"pRPLC_M2856","RAW_FILE_NAME":"nRPLC_M2856"}
},
{
"Subject ID":"-",
"Sample ID":"M2860",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H22","RAW_FILE_NAME":"pRPLC_M2860","RAW_FILE_NAME":"nRPLC_M2860"}
},
{
"Subject ID":"-",
"Sample ID":"M2939",
"Factors":{"Group":"Hypertophic cardiomyopathy"},
"Additional sample data":{"SampleID":"H26","RAW_FILE_NAME":"pRPLC_M2939","RAW_FILE_NAME":"nRPLC_M2939"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Cardiac tissue was excised and a mid-myocardial portion was used immediately for studies of mitochondrial respiration, or fixed in 4% paraformaldehyde (PFA) for paraffin embedding or in 4% PFA and 2% glutaraldehyde for TEM analysis. The remaining tissue was flash frozen in liquid nitrogen for all other assays.","SAMPLE_TYPE":"Heart"},

"TREATMENT":{"TREATMENT_SUMMARY":"N/A"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Sample Preparation. Roughly 30 mg of frozen heart tissue were homogenized in 500 µl ice-cold methanol by bead beating (MP bioscience cat# 6913-100, Solon, OH) at 4°C (2 x 45 s). Metabolites and complex lipids were extracted using a biphasic separation with cold methyl tert-butyl ether (MTBE), methanol and water. Briefly, 1 ml of ice-cold MTBE was added to 300 μl of the homogenate spiked-in with 40 µl deuterated lipid internal standards (Sciex, cat#: 5040156, lot#: LPISTDKIT-101). The samples were then sonicated (3 x 30 s) and agitated at 4°C for 30 min. After addition of 250 μl of ice-cold water, the samples were vortexed for 1 min and centrifuged at 14,000 g for 5 min at 20°C. The upper organic phase contains the lipids, the lower aqueous phase contains the metabolites and the proteins are precipitated at the bottom of the tube. For quality controls, 3 reference plasma samples (40 µl plasma) and 1 preparation blank were processed in parallel. 1) Metabolites: Proteins were further precipitated by adding 700 μl of 33/33/33 acetone/acetonitrile/methanol spiked-in with 15 labeled metabolite internal standards to 300 μl of the aqueous phase and 200 μl of the lipid phase and incubating the samples overnight at -20°C. After centrifugation at 17,000 g for 10 min at 4°C, the metabolic extracts were dried down to completion and resuspended in 100 μl 50/50 methanol/water. 2) Complex lipids: 700 µl of the organic phase was dried down under a stream of nitrogen and resolubilized in 200 μl of methanol for storage at -20°C until analysis. The day of the analysis, samples were dried down, resuspended in 300 μl of 10 mM ammonium acetate in 90/10 methanol/toluene and centrifuged at 16,000 g for 5 min at 24°C."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Lipid extracts were also analyzed using an Ultimate 3000 RSLC system coupled with a Q Exactive mass spectrometer (Thermo Scientific, Waltham, MA) as previously described6. Each sample was run twice in positive and negative ionization modes. Lipids were separated using an Accucore C18 column 2.1 x 150 mm, 2.6 μm (Thermo Scientific) and mobile phase solvents consisted in 10 mM ammonium acetate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 10 mM ammonium acetate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The Q Exactive was equipped with a HESI-II prob","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000 RS","COLUMN_NAME":"Thermo Accucore 2.1 x 150 mm, 2.6 μm"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"LC-MS peak extraction, alignment, quantification and annotation was performed using LipidSearch software version 4.2 (Thermo Scientific). Only lipids present in >2/3 of the samples were kept for further analysis. Median normalization (excluding TAG and DAG) was applied to correct for differential starting material quantity. Missing values were imputed by drawing from a random distribution of low values in the corresponding sample. Lipids were identified by matching the precursor ion mass to a database and the experimental MS/MS spectra to a spectral library containing theoretical fragmentation spectra. The identity of cardiolipins, detected as [M-H]-, was manually validated by investigating individual MS/MS spectra. Lipid abundances were reported as spectral counts.","MS_RESULTS_FILE":"ST001887_AN003055_Results.txt UNITS:MS count (log2) Has m/z:Yes Has RT:Yes RT units:Minutes"}

}