{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/04/PXD005350/DissectFeatures_BHM.xlsx"],"Mzxml":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/04/PXD005350/BHMMitoPLRPSTriversa10ul.mzXML"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/04/PXD005350/TopPIC.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":{"citationCount":0,"reanalysisCount":0,"viewCount":296,"searchCount":0},"additional":{"labhead_mail":["Alain.vanGool@radboudumc.nl"],"submitter":["Hans Wessels"],"technology_type":["Mass Spectrometry","Top-down proteomics"],"software":["Not available"],"submitter_keywords":["Mitochondrion","Bovine","Intact","Top-down proteomics","Protein"],"full_dataset_link":["http://www.ebi.ac.uk/pride/archive/projects/PXD005350"],"tissue":["Heart"],"sample_protocol":["Bovine heart mitochondria were a kind gift of Dr. Ilka Wittig (Goethe University, Frankfurt am Main, Germany). Mitochondria were diluted to 10 g/l with 8M Urea 10mM Tris-HCl pH8 and left on ice for 30 minutes for protein extraction. Subsequently, the sample was centrifuged at 22000 g for 20 minutes at 4 °C. The supernatant was collected and acidified (0.1% v/v formic acid) prior to LC-MS/MS analysis."],"repository":["Pride"],"quantification_method":["Not available"],"modification":["No PTMs are included in the dataset"],"data_protocol":["LC-MS/MS analysis was performed using a nano-Advance UHPLC (Bruker Daltonics, Bremen, Germany) connected to a maXis 4G ETD system (Bruker Daltonics, Bremen, Germany) via a TriVersa nanoMate (Advion, Ithaca, USA). 10 µl sample was injected onto an Acclaim PepMap C4, 0.3 x 5mm trapping column (Thermo Fisher Scientific, Waltham, USA) using a flow rate of 10 µl / min for 3 minutes. Subsequently, proteins were separated using a PLRP-S, 8µm, 4000Å, 0.3 x 150mm column operated at 50°C (Bruker-Michrom, Bremen, Germany) using a 60 minutes linear gradient of 10-45% acetonitrile in 0.1% formic acid at 3µl/min. Drying gas flow and temperature were set to 3l/min and 200°C, respectively, and nebulizer gas pressure was set to 0.0 bar. MS acquisition rate was set to 1 Hz and data have been acquired over a 300-3700 m/z mass range. AutoMSn CID fragmentation experiments were performed at 1Hz spectral rate for the top 2 most intense precursor ions using 30 seconds dynamic exclusion. Internal mass calibration was performed using the HP-1222 lockmass calibrant (Agilent Technologies). LC-MS data processing Data Processing : LC-MS data were automatically processed (calibration, protein signal extraction with Dissect™, deconvolution and obtention of monoisotopic masses with SNAP™, charge state filtering, similarity filtering, export of deconvoluted monoisotopic masses with corresponding retention time and intensities) in Data Analysis 4.2 (Bruker Daltonik, Bremen, Germany). Singly charged compounds have been automatically excluded. Only the isotopically resolved compounds have been taken into account. LC-MS/MS data processing LC-MS/MS data were calibrated internally using Data Analysis 4.2 (Bruker Daltonik, Bremen, Germany) and proprietary BAF files were converted to mzXML files using CompassXport. Monoisotopic peak picking and charge deconvolution were performed by MsDeconvGui v0.8.0.7370 (REF) and saved as MSAlign+ input files. Subsequently, TopPIC software [Bioinformatics. 2016 Jul 16. pii: btw398. TopPIC: a software tool for top-down mass spectrometry-based proteoform identification and characterization. Kou Q, Xun L, Liu X] was used to match acquired MS/MS data against the UniProt Bos taurus database. Parameters used were number of combined spectra: 1, search type: target + decoy, fixed modifications: none, maximum number of unexpected modifications: 2, error tolerance: 15 ppm, cutoff type: E-value, cutoff value: 0.01, allowed N-terminal forms: none + NME + NME_Acetylation, maximum mass shift of modifications: 700 Da, E-value computation: lookup table, MIScore threshold: 0.45."],"omics_type":["Proteomics"],"labhead":["Alain J van Gool"],"instrument_platform":["maXis"],"submission_type":["PARTIAL"],"labhead_affiliation":["Radboud Proteomics Center, Radboud Center for Mitochondrial Medicine, Department of Laboratory Medicine, Radboudumc"],"species":["Bos Taurus (bovine)"],"publication":["28855124 Schmit PO, Vialaret J, Wessels HJCT, van Gool AJ, Lehmann S, Gabelle A, Wood J, Bern M, Paape R, Suckau D, Kruppa G, Hirtz C. Towards a routine application of Top-Down approaches for label-free discovery workflows. J Proteomics. 2018 175:12-26 10.1016/j.jprot.2017.08.003"],"submitter_mail":["hans.wessels@radboudumc.nl"],"curator_keywords":["Biological"],"submitter_affiliation":["Radboudumc"],"submitter_country":["Netherlands"],"pubmed_abstract":["Thanks to proteomics investigations, our vision of the role of different protein isoforms in the pathophysiology of diseases has largely evolved. The idea that protein biomarkers like tau, amyloid peptides, ApoE, cystatin, or neurogranin are represented in body fluids as single species is obviously over-simplified, as most proteins are present in different isoforms and subjected to numerous processing and post-translational modifications. Measuring the intact mass of proteins by MS has the advantage to provide information on the presence and relative amount of the different proteoforms. Such Top-Down approaches typically require a high degree of sample pre-fractionation to allow the MS system to deliver optimal performance in terms of dynamic range, mass accuracy and resolution. In clinical studies, however, the requirements for pre-analytical robustness and sample size large enough for statistical power restrict the routine use of a high degree of sample pre-fractionation. In this study, we have investigated the capacities of current-generation Ultra-High Resolution Q-Tof systems to deal with high complexity intact protein samples and have evaluated the approach on a cohort of patients suffering from neurodegenerative disease. Statistical analysis has shown that several proteoforms can be used to distinguish Alzheimer disease patients from patients suffering from other neurodegenerative disease.