title: Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts frommdx-4cvSkeletal Muscles.
abstract: The full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normalversusdystrophicmdx-4cvskeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that themdx-4cvmouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen inmdx-4cvmuscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context.
authors.affiliation: Department of Biology, Maynooth University, National University of Ireland, Maynooth Co. Kildare, Ireland. sandra.murphy@nuim.ie.
authors.forename: Sandra
authors.initials: S
authors.lastname: Murphy
authors.affiliation: Department of Physiology II, University of Bonn, Bonn D-53115, Germany. margit.zweyer@ukb.uni-bonn.de.
authors.forename: Margit
authors.initials: M
authors.lastname: Zweyer
authors.affiliation: Department of Physiology II, University of Bonn, Bonn D-53115, Germany. mundegar@yahoo.de.
authors.forename: Rustam R
authors.initials: RR
authors.lastname: Mundegar
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland. michael.henry@dcu.ie.
authors.forename: Michael
authors.initials: M
authors.lastname: Henry
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland. paula.meleady@dcu.ie.
authors.forename: Paula
authors.initials: P
authors.lastname: Meleady
authors.affiliation: Department of Physiology II, University of Bonn, Bonn D-53115, Germany. dieter.swandulla@ukb.uni-bonn.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Swandulla
authors.affiliation: Department of Biology, Maynooth University, National University of Ireland, Maynooth Co. Kildare, Ireland. kay.ohlendieck@nuim.ie.
authors.forename: Kay
authors.initials: K
authors.lastname: Ohlendieck
keywords: Dp427; Duchenne muscular dystrophy; collagen; dystrophin; myofibrosis