#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