On-Membrane Digestion Technology for Muscle Proteomics

Ohlendieck,

doi:10.6000/1929-6037.2013.02.01.1

Cited by 7 publications

(9 citation statements)

References 137 publications

(155 reference statements)

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“…To remove cross-contaminating subcellular structures derived from transverse tubules and the highly abundant sarcoplasmic reticulum, lectin affinity agglutination and mild detergent washing was employed to isolate sarcolemma vesicles. Since standard two-dimensional gel electrophoresis and in-gel digestion had failed to identify the dystrophin-glycoprotein complex during early proteomic studies of muscular dystrophy [23] , [25] , a combination of one-dimensional gradient gel electrophoresis, electrophoretic protein transfer and on-membrane digestion were applied prior to mass spectrometric analysis [86] .…”

Section: Proteomics Of the Dystrophin Complexomementioning

confidence: 99%

The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle

Murphy

Ohlendieck

2016

Computational and Structural Biotechnology Journal

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The development of advanced mass spectrometric methodology has decisively enhanced the analytical capabilities for studies into the composition and dynamics of multi-subunit protein complexes and their associated components. Large-scale complexome profiling is an approach that combines the systematic isolation and enrichment of protein assemblies with sophisticated mass spectrometry-based identification methods. In skeletal muscles, the membrane cytoskeletal protein dystrophin of 427 kDa forms tight interactions with a variety of sarcolemmal, cytosolic and extracellular proteins, which in turn associate with key components of the extracellular matrix and the intracellular cytoskeleton. A major function of this enormous assembly of proteins, including dystroglycans, sarcoglycans, syntrophins, dystrobrevins, sarcospan, laminin and cortical actin, is postulated to stabilize muscle fibres during the physical tensions of continuous excitation-contraction-relaxation cycles. This article reviews the evidence from recent proteomic studies that have focused on the characterization of the dystrophin-glycoprotein complex and its central role in the establishment of the cytoskeleton-sarcolemma-matrisome axis. Proteomic findings suggest a close linkage of the core dystrophin complex with a variety of protein species, including tubulin, vimentin, desmin, annexin, proteoglycans and collagens. Since the almost complete absence of dystrophin is the underlying cause for X-linked muscular dystrophy, a more detailed understanding of the composition, structure and plasticity of the dystrophin complexome may have considerable biomedical implications.

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Section: Proteomics Of the Dystrophin Complexomementioning

confidence: 99%

The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle

Murphy

Ohlendieck

2016

Computational and Structural Biotechnology Journal

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“…However, GE methods can routinely detect fragments of these high-molecular-mass proteins. Many integral or membrane-associated muscle proteins are under-represented in 2D gels and their detailed proteomic analysis has to be carried out with enrichment methods prior to 2D-GE analysis, supplementing LC methods and/or alternative 1D gradient gel systems [99,100,101,102]. The most highly abundant proteins in muscle homogenates are ACTs, MyHCs, MLCs, TMs and TNs.…”

Section: Cataloguing Of the Skeletal Muscle Proteome Using Two-dimmentioning

confidence: 99%

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

Murphy¹,

Dowling²,

Ohlendieck³

2016

Proteomes

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The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins.

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“…Label-free mass spectrometry has also been successfully employed to compare normal versus stressed muscle specimens and identi ed distinct changes in HSP molecules [70,125,126]. Since skeletal muscles contain some of the largest proteins in the human body, such as nebulin, obscurin and titin, alternative onmembrane digestion approaches have been developed to study these giant proteins [159][160][161]. In future studies, gradient gel electrophoretic separation in combination with on- Table 4.…”

Section: Molecular Chaperonementioning

confidence: 99%

Proteomic profiling of the HSPB chaperonome: Mass spectrometric identification of small heat shock proteins in stressed skeletal muscles

Murphy¹,

Ohlendieck

2015

JIOMICS

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e continuing maintenance of protein homeostasis and the protection of proteomic integrity is essential for the survival of complex cellular systems under stressful conditions. Proteostasis is maintained by a complex system of protective pathways that involve several classes of molecular chaperones, now referred to as the chaperonome. e elaborate interplay of these components averts detrimental protein aggregation and supports proteins in resuming their functional fold. In skeletal muscle tissues, molecular chaperones protect contractile functions throughout bre adaptations to changed physiological demands and prevent tissue damage during acute phases of protein misfolding or prolonged periods of harmful protein accumulation. is results in considerable changes in the expression pro le of individual members of the large family of heat shock proteins. Systematic proteomic surveys of skeletal muscle tissues have revealed that the concentration of small heat shock proteins is especially a ected following strenuous exercise, in various neuromuscular disorders and during the natural aging process. Of the 10 identi ed members of the small heat shock protein HSPB family, HSPB1 (Hsp25), HSPB2 (MKBP), HSPB3 (Hsp27), HSPB4 (αA-crystallin), HSPB5 (αB-crystallin), HSPB6 (Hsp20), HSPB7 (cardiovascular cvHsp) and HSPB8 (Hsp22) are clearly present in skeletal muscle tissues. is review outlines the proteomic identi cation of small heat shock proteins and their muscle-speci c expression and induction patterns in health and disease. Since HSPB molecules are of relatively low molecular mass, belong to the markedly soluble type of proteins and represent critical pro-survival proteins that are intrinsically involved in the prevention of stress-induced bre damage, they present ideal muscle-associated biomarker candidates for the establishment of superior diagnostic and therapy-monitoring approaches to assess stress-related skeletal muscle degeneration.

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On-Membrane Digestion Technology for Muscle Proteomics

Cited by 7 publications

References 137 publications

The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle

The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

Proteomic profiling of the HSPB chaperonome: Mass spectrometric identification of small heat shock proteins in stressed skeletal muscles

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