#METABOLOMICS WORKBENCH kcontrep_20210713_090240 DATATRACK_ID:2744 STUDY_ID:ST001887 ANALYSIS_ID:AN003055 PROJECT_ID:000000 VERSION 1 CREATED_ON July 30, 2021, 1:00 pm #PROJECT PR:PROJECT_TITLE Multi-omics study of hypertrophic cardiomyopathy PR:PROJECT_SUMMARY Multi-omics study of human heart tissues in the context of hypertrophic PR:PROJECT_SUMMARY cardiomyopathy PR:INSTITUTE Stanford University PR:LAST_NAME Contrepois PR:FIRST_NAME Kevin PR:ADDRESS 300 Pasteur Dr PR:EMAIL kcontrep@stanford.edu PR:PHONE 6506664538 #STUDY ST:STUDY_TITLE Untargeted lipidomics of hypertrophic cardiomyopathy (part II) ST:STUDY_SUMMARY Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the ST:STUDY_SUMMARY effects of individual gene variants on sarcomeric protein biomechanics. At the ST:STUDY_SUMMARY cellular level, HCM mutations most commonly enhance force production, leading to ST:STUDY_SUMMARY higher energy demands. Despite significant advances in elucidating sarcomeric ST:STUDY_SUMMARY structure-function relationships, there is still much to be learned about the ST:STUDY_SUMMARY mechanisms that link altered cardiac energetics to HCM phenotypes. In this work, ST:STUDY_SUMMARY we test the hypothesis that changes in cardiac energetics represent a common ST:STUDY_SUMMARY pathophysiologic pathway in HCM. ST:INSTITUTE Stanford University ST:LAST_NAME Contrepois ST:FIRST_NAME Kevin ST:ADDRESS 300 Pasteur Dr ST:EMAIL kcontrep@stanford.edu ST:PHONE 6506664538 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - D1234 Group:Donor SampleID=D1; RAW_FILE_NAME=pRPLC_D1234; RAW_FILE_NAME=nRPLC_D1234 SUBJECT_SAMPLE_FACTORS - D1331 Group:Donor SampleID=D3; RAW_FILE_NAME=pRPLC_D1331; RAW_FILE_NAME=nRPLC_D1331 SUBJECT_SAMPLE_FACTORS - D2507 Group:Donor SampleID=D8; RAW_FILE_NAME=pRPLC_D2507; RAW_FILE_NAME=nRPLC_D2507 SUBJECT_SAMPLE_FACTORS - D2540 Group:Donor SampleID=D9; RAW_FILE_NAME=pRPLC_D2540; RAW_FILE_NAME=nRPLC_D2540 SUBJECT_SAMPLE_FACTORS - D2552 Group:Donor SampleID=D10; RAW_FILE_NAME=pRPLC_D2552; RAW_FILE_NAME=nRPLC_D2552 SUBJECT_SAMPLE_FACTORS - D2554 Group:Donor SampleID=D11; RAW_FILE_NAME=pRPLC_D2554; RAW_FILE_NAME=nRPLC_D2554 SUBJECT_SAMPLE_FACTORS - M433 Group:Hypertophic cardiomyopathy SampleID=H4; RAW_FILE_NAME=pRPLC_M433; RAW_FILE_NAME=nRPLC_M433 SUBJECT_SAMPLE_FACTORS - M467 Group:Hypertophic cardiomyopathy SampleID=H5; RAW_FILE_NAME=pRPLC_M467; RAW_FILE_NAME=nRPLC_M467 SUBJECT_SAMPLE_FACTORS - M1385 Group:Hypertophic cardiomyopathy SampleID=H6; RAW_FILE_NAME=pRPLC_M1385; RAW_FILE_NAME=nRPLC_M1385 SUBJECT_SAMPLE_FACTORS - M1455 Group:Hypertophic cardiomyopathy SampleID=H7; RAW_FILE_NAME=pRPLC_M1455; RAW_FILE_NAME=nRPLC_M1455 SUBJECT_SAMPLE_FACTORS - M2622 Group:Hypertophic cardiomyopathy SampleID=H8; RAW_FILE_NAME=pRPLC_M2622; RAW_FILE_NAME=nRPLC_M2622 SUBJECT_SAMPLE_FACTORS - M2673 Group:Hypertophic cardiomyopathy SampleID=H11; RAW_FILE_NAME=pRPLC_M2673; RAW_FILE_NAME=nRPLC_M2673 SUBJECT_SAMPLE_FACTORS - M2692 Group:Hypertophic cardiomyopathy SampleID=H13; RAW_FILE_NAME=pRPLC_M2692; RAW_FILE_NAME=nRPLC_M2692 SUBJECT_SAMPLE_FACTORS - M2799 Group:Hypertophic cardiomyopathy SampleID=H16; RAW_FILE_NAME=pRPLC_M2799; RAW_FILE_NAME=nRPLC_M2799 SUBJECT_SAMPLE_FACTORS - M2800 Group:Hypertophic cardiomyopathy SampleID=H17; RAW_FILE_NAME=pRPLC_M2800; RAW_FILE_NAME=nRPLC_M2800 SUBJECT_SAMPLE_FACTORS - M2803 Group:Hypertophic cardiomyopathy SampleID=H18; RAW_FILE_NAME=pRPLC_M2803; RAW_FILE_NAME=nRPLC_M2803 SUBJECT_SAMPLE_FACTORS - M2856 Group:Hypertophic cardiomyopathy SampleID=H21; RAW_FILE_NAME=pRPLC_M2856; RAW_FILE_NAME=nRPLC_M2856 SUBJECT_SAMPLE_FACTORS - M2860 Group:Hypertophic cardiomyopathy SampleID=H22; RAW_FILE_NAME=pRPLC_M2860; RAW_FILE_NAME=nRPLC_M2860 SUBJECT_SAMPLE_FACTORS - M2939 Group:Hypertophic cardiomyopathy SampleID=H26; RAW_FILE_NAME=pRPLC_M2939; RAW_FILE_NAME=nRPLC_M2939 #COLLECTION CO:COLLECTION_SUMMARY Cardiac tissue was excised and a mid-myocardial portion was used immediately for CO:COLLECTION_SUMMARY studies of mitochondrial respiration, or fixed in 4% paraformaldehyde (PFA) for CO:COLLECTION_SUMMARY paraffin embedding or in 4% PFA and 2% glutaraldehyde for TEM analysis. The CO:COLLECTION_SUMMARY remaining tissue was flash frozen in liquid nitrogen for all other assays. CO:SAMPLE_TYPE Heart #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sample Preparation. Roughly 30 mg of frozen heart tissue were homogenized in 500 SP:SAMPLEPREP_SUMMARY µl ice-cold methanol by bead beating (MP bioscience cat# 6913-100, Solon, OH) SP:SAMPLEPREP_SUMMARY at 4°C (2 x 45 s). Metabolites and complex lipids were extracted using a SP:SAMPLEPREP_SUMMARY biphasic separation with cold methyl tert-butyl ether (MTBE), methanol and SP:SAMPLEPREP_SUMMARY water. Briefly, 1 ml of ice-cold MTBE was added to 300 μl of the homogenate SP:SAMPLEPREP_SUMMARY spiked-in with 40 µl deuterated lipid internal standards (Sciex, cat#: 5040156, SP:SAMPLEPREP_SUMMARY lot#: LPISTDKIT-101). The samples were then sonicated (3 x 30 s) and agitated at SP:SAMPLEPREP_SUMMARY 4°C for 30 min. After addition of 250 μl of ice-cold water, the samples were SP:SAMPLEPREP_SUMMARY vortexed for 1 min and centrifuged at 14,000 g for 5 min at 20°C. The upper SP:SAMPLEPREP_SUMMARY organic phase contains the lipids, the lower aqueous phase contains the SP:SAMPLEPREP_SUMMARY metabolites and the proteins are precipitated at the bottom of the tube. For SP:SAMPLEPREP_SUMMARY quality controls, 3 reference plasma samples (40 µl plasma) and 1 preparation SP:SAMPLEPREP_SUMMARY blank were processed in parallel. 1) Metabolites: Proteins were further SP:SAMPLEPREP_SUMMARY precipitated by adding 700 μl of 33/33/33 acetone/acetonitrile/methanol SP:SAMPLEPREP_SUMMARY spiked-in with 15 labeled metabolite internal standards to 300 μl of the SP:SAMPLEPREP_SUMMARY aqueous phase and 200 μl of the lipid phase and incubating the samples SP:SAMPLEPREP_SUMMARY overnight at -20°C. After centrifugation at 17,000 g for 10 min at 4°C, the SP:SAMPLEPREP_SUMMARY metabolic extracts were dried down to completion and resuspended in 100 μl SP:SAMPLEPREP_SUMMARY 50/50 methanol/water. 2) Complex lipids: 700 µl of the organic phase was dried SP:SAMPLEPREP_SUMMARY down under a stream of nitrogen and resolubilized in 200 μl of methanol for SP:SAMPLEPREP_SUMMARY storage at -20°C until analysis. The day of the analysis, samples were dried SP:SAMPLEPREP_SUMMARY down, resuspended in 300 μl of 10 mM ammonium acetate in 90/10 methanol/toluene SP:SAMPLEPREP_SUMMARY and centrifuged at 16,000 g for 5 min at 24°C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Lipid extracts were also analyzed using an Ultimate 3000 RSLC system coupled CH:CHROMATOGRAPHY_SUMMARY with a Q Exactive mass spectrometer (Thermo Scientific, Waltham, MA) as CH:CHROMATOGRAPHY_SUMMARY previously described6. Each sample was run twice in positive and negative CH:CHROMATOGRAPHY_SUMMARY ionization modes. Lipids were separated using an Accucore C18 column 2.1 x 150 CH:CHROMATOGRAPHY_SUMMARY mm, 2.6 μm (Thermo Scientific) and mobile phase solvents consisted in 10 mM CH:CHROMATOGRAPHY_SUMMARY ammonium acetate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 10 mM CH:CHROMATOGRAPHY_SUMMARY ammonium acetate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The CH:CHROMATOGRAPHY_SUMMARY Q Exactive was equipped with a HESI-II prob CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Thermo Accucore 2.1 x 150 mm, 2.6 μm #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 LC-MS peak extraction, alignment, quantification and annotation was performed MS:MS_COMMENTS using LipidSearch software version 4.2 (Thermo Scientific). Only lipids present MS:MS_COMMENTS in >2/3 of the samples were kept for further analysis. Median normalization MS:MS_COMMENTS (excluding TAG and DAG) was applied to correct for differential starting MS:MS_COMMENTS material quantity. Missing values were imputed by drawing from a random MS:MS_COMMENTS distribution of low values in the corresponding sample. Lipids were identified MS:MS_COMMENTS by matching the precursor ion mass to a database and the experimental MS/MS MS:MS_COMMENTS spectra to a spectral library containing theoretical fragmentation spectra. The MS:MS_COMMENTS identity of cardiolipins, detected as [M-H]-, was manually validated by MS:MS_COMMENTS investigating individual MS/MS spectra. Lipid abundances were reported as MS:MS_COMMENTS spectral counts. MS:MS_RESULTS_FILE ST001887_AN003055_Results.txt UNITS:MS count (log2) Has m/z:Yes Has RT:Yes RT units:Minutes #END