{"exp_accession":"CRX8","title":"Complexome analysis of mitochondria of fibroblasts from Barth syndrome patients carrying mutations in the TAZ gene.","description":"To study whether there would be changes at the mitochondrial protein levels in Barth syndrome (BTHS) patients' fibroblasts, we studied skin fibroblasts from four patients carrying different mutated versions of the tafazzin gene (P1: c.153C > G, P2: c.239-1G > A, P3: c.170G > T, P4: c.110-1G > C) and performed complexome profiling in comparison to a healthy control. This method allows analyzing changes in the composition and abundance of mitochondrial complexes based on the migration profiles of their constituent proteins obtained by mass spectrometry after separation by blue native electrophoresis.","sample_processing":"Primary skin fibroblasts from four human BTHS patients (P1: TAZ001, P2: TAZ002, P3: TAZ006 and P4: TAZ013) and a healthy control (C103T) were obtained according to institutional guidelines and identifiable clinical and personal data from the patients were not available for this study. All patients were diagnosed with BTHS on the basis of ML(CL) analysis. Cells were cultured in DMEM or Ham's F-10 with l-glutamine (Bio-Whittaker) growth media, supplemented with 10% fetal bovine serum (Bio-Whittaker), 25 mM HEPES buffer (Bio-Whittaker), 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies), and 250 ng/ml Fungizone (Life Technologies) in a humidified atmosphere with 5% CO2 at 37 °C. Cell pellets were resuspended in 125 mM sucrose, 1 mM EDTA, 20 mM Tris/HCl pH 7.4 and disrupted by 10 strokes in a glass/Teflon Potter-Elvehjem homogenizer. The homogenates were mixed with 875 mM sucrose, 1 mM EDTA, 20 mM Tris/HCL pH 7.4 to adjust the sucrose concentration to 250 mM and centrifuged at 1000g for 10 min at 4 °C. Supernatants were centrifuged at 14000g for 10 min at 4 °C. The mitochondrial pellets were resuspended in 250 mM sucrose, 1 mM EDTA, 20 mM Tris/HCl pH 7.4. Protein concentration was determined by the Lowry method. The gel pieces were incubated for 60 min in buffer containing 5 mM dithiothreitol,50 mM ammonium hydrogen carbonate (AHC) followed by incubation in 15 mM 2-chloroacetamide, 50 mM AHC for 45 min. Gel slices were then washed with 50% methanol, 50 mM AHC for 15 min and air-dried for 45 min. The dehydrated gel pieces were swollen in 20μl5ng/μltryp-sin (Promega, V5111), 50 mM AHC, 1 mM CaCl2 for 30 min at 4°C.After addition of 50μl 50 mM AHC, the plates were sealed and incubated overnight at 37 °C for tryptic digest. The peptides were eluted by centrifugation into a 96-well PCR microplate and the remaining peptides were extracted twice in 50μl 30% acetonitrile, 3% formic acid. The supernatants were dried in a SpeedVac concentrator and the peptides resuspended in 20μl 5% acetonitrile, 0.5% formic acid.","data_processing":"The analysis of the RAW files was performed using the MaxQuant software package (version 1.4.1.2 and 1.5.0.25, www.maxquant.org (Cox & Mann, 2008)). The extracted spectra were matched against the reviewed H. sapiens NCBI RefSeq database release 55. Sequences of known contaminants were added to this database and the reverse decoy was strictly set to FDR of 0.01. Database searches were done with 20 ppm and 0.5 Da mass tolerances for precursor ions and fragmented ions, respectively. Trypsin (two missed cleavages allowed) was selected as the protease. Dynamic modifications included N-terminal acetylation and oxidation of methionine. Cystein carbamidomethylation was set as a fixed modification. Keratins, hemoglobins and trypsin were removed from the list. Protein abundances were determined by label-free quantitation using the composite IBAQ intensity values determined by MaxQuant (Cox & Mann, 2008) and normalized within single or multiple migration profiles of individual proteins. The profiles were hierarchically clustered by distance measures based on Pearson correlation coefficient (uncentered) and the average linkage method and further analyzed by manual correlation profiling. Mass scales of the profiles for soluble and integral membrane proteins were determined as previously described using known protein complexes as standards(Heide et al., 2012). For comparison between the different samples, the total protein abundance (IBAQ) values were corrected for variations between the different profiles and then, the maximum abundance found in any of the profiles of the different samples was set to one for each individual protein. The clustering and the visualization and analysis of the heat maps were done with the NOVA software v0.5 (Giese et al., 2014) and MS Excel.","separation_method":{"term_name":"blue native polyacrylamide gel electrophoresis","term_code":"CHMO:0002276"},"MS_instrument":{"term_name":"Q Exactive","term_code":"MS:1001911"},"MS_instrument_details":"The tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in a Thermo Scientific Q Exactive 2.0 Orbitrap Mass Spectrometry System equipped with a nano-flow high performance liquid chromatography system Easy nLC-1000 at the front end and the Thermo Scientific Xcalibur 2.2 SP1 Software Package. Peptides were separated on 3μm reprosil-pur C18 beads (Dr Maisch GmbH, Germany) filled into a PicoTip emitter column (8 ±1μm Silica Tip; New Objectives, USA) in 40 min HPLC runs using a 30 min gradient of 5% to 35% acetonitrile with 0.1% formic acid, followed by a column wash with 80% acetonitrile and re-equilibration with 5%acetonitrile for 5 min each. Peptides were analyzed on-line in positive mode by a mass spectrometry method programmed to fragment the top 20 most abundant peptides. Full scan MS mode (400 to 1400 m/z) was operated at a resolution of 70 000 with automatic gain control (AGC) target of 1×10e6 ions and a maximum ion transfer of 20 ms. Selected ions for MS/MS were analyzed using the following parameters: resolution 17,500; AGC target of 1 × 10e5; maximum ion transfer of 50 ms; 4.0 m/z isolation window; for CID a normalized collision energy 30% was used; and dynamic exclusion of 30.0 s. A lock mass ion (m/z =445.12) was used for internal calibration.","quantification":{"term_name":"label-free proteingroup level quantitation","term_code":"MS:1002022"},"protein_identifier":{"term_name":"GI number (protein)","term_code":"data:2327"},"publication":"Chatzispyrou, Iliana A., et al. \"Barth syndrome cells display widespread remodeling of  mitochondrial complexes without affecting metabolic flux distribution.\" Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1864.11 (2018): 3650-3658.","publication_doi":null,"publication_ref":"Chatzispyrou et al., 2018","publication_pmid":30251684,"submitter":{"usr_accession":"SUB8","affiliation":"CMBI, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands","user":{"first_name":"Joeri","last_name":"van Strien"}},"sample_count":5,"samples":[{"smp_accession":"CRS21","name":"TAZ013","organism_part":[{"term_name":"fibroblast","term_code":"BTO:0000452"}],"condition":[{"term_name":"disease","term_code":"EFO:0000408"}],"cell_compartment":[{"term_name":"mitochondrion","term_code":"GO:0005739"}],"experiment":"CRX8","protein_file":{"id":12,"file_type":"Protein","name":"protein_abundances_all","description":"contains protein-level abundance profiles for all samples, as iBAQ values.  The coverage column contains Unique + razor sequence coverage.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_protein_abundances.ctab"},"peptide_file":null,"processed_file":{"id":13,"file_type":"Processed","name":"annotated_abundances_all","description":"contains the same protein-level abundances as in comptab file, but includes columns with additional information, like protein names, gene symbols etc. Stored in plain text csv format.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_annot_abundances.csv"},"gel":"CRP8","created_at":"2020-06-12T13:35:40.408818Z","updated_at":"2020-06-20T11:30:55.332285Z","species":[{"term_name":"Homo sapiens","term_code":"NCBITaxon:9606"}]},{"smp_accession":"CRS20","name":"TAZ006","organism_part":[{"term_name":"fibroblast","term_code":"BTO:0000452"}],"condition":[{"term_name":"disease","term_code":"EFO:0000408"}],"cell_compartment":[{"term_name":"mitochondrion","term_code":"GO:0005739"}],"experiment":"CRX8","protein_file":{"id":12,"file_type":"Protein","name":"protein_abundances_all","description":"contains protein-level abundance profiles for all samples, as iBAQ values.  The coverage column contains Unique + razor sequence coverage.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_protein_abundances.ctab"},"peptide_file":null,"processed_file":{"id":13,"file_type":"Processed","name":"annotated_abundances_all","description":"contains the same protein-level abundances as in comptab file, but includes columns with additional information, like protein names, gene symbols etc. Stored in plain text csv format.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_annot_abundances.csv"},"gel":"CRP8","created_at":"2020-06-12T13:35:40.339700Z","updated_at":"2020-06-20T11:30:36.395753Z","species":[{"term_name":"Homo sapiens","term_code":"NCBITaxon:9606"}]},{"smp_accession":"CRS19","name":"TAZ002","organism_part":[{"term_name":"fibroblast","term_code":"BTO:0000452"}],"condition":[{"term_name":"disease","term_code":"EFO:0000408"}],"cell_compartment":[{"term_name":"mitochondrion","term_code":"GO:0005739"}],"experiment":"CRX8","protein_file":{"id":12,"file_type":"Protein","name":"protein_abundances_all","description":"contains protein-level abundance profiles for all samples, as iBAQ values.  The coverage column contains Unique + razor sequence coverage.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_protein_abundances.ctab"},"peptide_file":null,"processed_file":{"id":13,"file_type":"Processed","name":"annotated_abundances_all","description":"contains the same protein-level abundances as in comptab file, but includes columns with additional information, like protein names, gene symbols etc. Stored in plain text csv format.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_annot_abundances.csv"},"gel":"CRP8","created_at":"2020-06-12T13:35:40.271746Z","updated_at":"2020-06-20T11:30:14.801936Z","species":[{"term_name":"Homo sapiens","term_code":"NCBITaxon:9606"}]},{"smp_accession":"CRS18","name":"TAZ001","organism_part":[{"term_name":"fibroblast","term_code":"BTO:0000452"}],"condition":[{"term_name":"disease","term_code":"EFO:0000408"}],"cell_compartment":[{"term_name":"mitochondrion","term_code":"GO:0005739"}],"experiment":"CRX8","protein_file":{"id":12,"file_type":"Protein","name":"protein_abundances_all","description":"contains protein-level abundance profiles for all samples, as iBAQ values.  The coverage column contains Unique + razor sequence coverage.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_protein_abundances.ctab"},"peptide_file":null,"processed_file":{"id":13,"file_type":"Processed","name":"annotated_abundances_all","description":"contains the same protein-level abundances as in comptab file, but includes columns with additional information, like protein names, gene symbols etc. Stored in plain text csv format.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_annot_abundances.csv"},"gel":"CRP8","created_at":"2020-06-12T13:35:40.203926Z","updated_at":"2020-06-20T11:29:44.520411Z","species":[{"term_name":"Homo sapiens","term_code":"NCBITaxon:9606"}]},{"smp_accession":"CRS17","name":"C103T","organism_part":[{"term_name":"fibroblast","term_code":"BTO:0000452"}],"condition":[{"term_name":"control","term_code":"EFO:0001461"}],"cell_compartment":[{"term_name":"mitochondrion","term_code":"GO:0005739"}],"experiment":"CRX8","protein_file":{"id":12,"file_type":"Protein","name":"protein_abundances_all","description":"contains protein-level abundance profiles for all samples, as iBAQ values.  The coverage column contains Unique + razor sequence coverage.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_protein_abundances.ctab"},"peptide_file":null,"processed_file":{"id":13,"file_type":"Processed","name":"annotated_abundances_all","description":"contains the same protein-level abundances as in comptab file, but includes columns with additional information, like protein names, gene symbols etc. Stored in plain text csv format.","data_file":"https://www3.cmbi.umcn.nl/cedar/media/sampledata/TAZ_annot_abundances.csv"},"gel":"CRP8","created_at":"2020-06-12T13:35:40.012900Z","updated_at":"2020-06-20T11:29:18.340261Z","species":[{"term_name":"Homo sapiens","term_code":"NCBITaxon:9606"}]}],"created_at":"2020-06-12T13:35:39.557254Z","updated_at":"2020-06-28T13:04:40.888033Z","protxchange_id":null}