Although the SERCA proteins are encoded by three genes, of which the SERCA1 and SERCA2 genes are expressed in voluntary fibres, the isoform diversity of this abundant ion pump is drastically increased by alternative splicing of the transcripts and various posttranslational modifications [ 38 ], producing more than 10 different SERCA isoforms [ 39 ].
These cellular processes significantly increase muscle protein diversity. Biological hierarchy of the neuromuscular system. Shown are the organisation of the genome, transcriptome and proteome of motor neurons and skeletal muscles.
The histological image illustrates neuromuscular junctions on individual muscle fibres labelled for the presence of the enzyme acetylcholinesterase. The wide and dynamic expression range of proteins within a specific tissue makes it impossible to separate and detect all protein species with currently available biochemical techniques. In addition, most tissue types are heterogeneous in composition. Aside from the main contractile cells of differing contractile properties, such as slow oxidative type I , moderately fast oxidative glycolytic type IIa and fast glycolytic type IIb fibres, muscle contains extended layers of connective tissues, capillaries and nerve cells [ 40 — 42 ].
Thus the starting material for almost all invasive biochemical studies, that is, homogenised muscle tissues, contains a certain degree of cell types that have originated from the tendon, epimysium, endomysium, perimysium, muscle spindles, satellite cells, blood vessels and motor neurons. This analytical fact has to be taken into account when one interprets proteomic findings from total tissue extracts. In the past, microassay systems have been developed to study single-fibre preparations biochemically [ 43 ], so it might be feasible to investigate such cellular preparations by using miniaturised separation protocols and proteomic techniques with enhanced sensitivity in the future.
Concentration differences between highly abundant muscle proteins, such as glycolytic enzymes, and low-abundance muscle proteins, such as surface signaling receptors, have been estimated to be several orders of magnitude. Standard gel electrophoretic or liquid chromatographic methods are not capable of separating this vast range of muscle proteins with differing densities. Besides the biological fact that it is extremely difficult to accurately define a fixed protein complement in highly adaptable and dynamic fibre populations as 'the skeletal muscle proteome', the most obvious technical obstacle to the study of entire muscle proteomes is the limited availability of all-encompassing protein analytical capabilities.
Currently no one set of biochemical techniques exists that can efficiently separate and consistently detect the total protein complement of a given cell type. Hence, with respect to interpreting findings from analytical gel electrophoresis, it is important to take into account various technical limitations. Two-dimensional gel electrophoresis of crude tissue extracts usually underestimates the presence of low abundance elements, the amount of integral membrane proteins and components with very high molecular masses in complex tissues [ 19 , 44 — 46 ]. In addition, proteins with extreme p I values are often not properly resolved at the edge of large gel systems with a wide p I range.
This problem can be partially addressed by using narrow-range p I gels in the first dimension or by employing overlapping gel systems covering several p I ranges [ 44 ]. Another important issue in two-dimensional gel electrophoresis is the distortion of protein spots due to abundant protein species or the presence of isforms with extensive posttranslational modifications [ 45 ]. For example, high levels of heterogeneous glycosylation patterns can result in broad or overlapping spot patterns, which are difficult to pick for in-gel digestion procedures.
Importantly, the presence of muscle proteins with a high density, such as myosin heavy chains, myosin light chains, troponins, tropomyosins and actins, can distort certain zones within the two-dimensional separation pattern and thus potentially contaminate other protein spots. In such a case, the densitometric analysis of a specific spot and the identification of the most abundant protein species present in this gel region might not perfectly correlate.
Proteolytic degradation products of high molecular mass proteins may also complicate the analysis of the gel image. However, despite these technical limitations, gel electrophoresis-based proteomics results in excellent coverage of soluble and abundant muscle proteins involved in the regulation and execution of the contraction-relaxation cycle, energy metabolism and the cellular stress response [ 13 ]. The use of crude tissue extracts as starting material has the advantage of representing the entire soluble protein complement without the potential danger of protein desorption or artefactual entrapment by complex separation steps.
On the other hand, organelle and membrane proteomics reduces sample complexity by focusing on distinct subsets of protein populations, as outlined below. Ideally, proteomic studies of skeletal muscle tissues should first use both crude extracts and distinct subcellular fractions as starting material and second employ gel electrophoresis and liquid chromatography in parallel for the separation of as many different classes of muscle proteins as possible.
The long-term goal of proteomic profiling studies is the cataloguing of all expressed protein species and the establishment of comprehensive biomarker signatures that characterise physiological processes during development and natural aging, as well as disease progression in common pathologies. The flowchart in Figure 2 outlines the main steps of proteomic profiling studies.
Proteomic profiling of skeletal muscle. The flowchart outlines the various preparative and analytical steps involved in the routine mass spectrometry-based proteomic investigation of contractile tissues. Over the past few years, mass spectrometry-based proteomics has successfully catalogued several hundred of the most abundant and soluble muscle-associated protein species and identified several thousand distinct protein isoforms present in skeletal muscle tissues [ 47 — 50 ].
Muscle proteomics has been applied to the comprehensive biochemical profiling of developing, maturing and aging muscle [ 51 — 56 ], as well as the analysis of contractile tissues undergoing physiological adaptations seen in disuse atrophy, physical exercise and chronic muscle transformation [ 57 — 63 ]. Biomedical investigations into proteome-wide alterations in skeletal muscle tissues were also used to establish novel biomarker signatures of neuromuscular pathologies.
Disease-specific markers were determined for muscle-associated diseases such as dystrophinopathy [ 64 — 66 ], dysferlinopathy [ 67 ], traumatic denervation [ 68 ], obesity [ 69 ], diabetes-related contractile weakness [ 70 ], sepsis [ 71 ], hypokalemic myopathy [ 72 ], inclusion body myositis [ 73 ] and reducing body myopathy [ 74 ]. Since skeletal muscle biology is highly relevant to the meat industry, muscle proteomics has been widely applied to cataloguing and studying protein complements in livestock [ 75 — 77 ].
These studies have especially focused on the proteomic evaluation of hypertrophy in chicken [ 78 , 79 ], sheep [ 80 ], pig [ 81 — 83 ] and cow [ 84 ] muscles. Table 1 lists key findings from recent proteomic studies focusing on the biochemical characterisation of skeletal muscle tissues. Biomarker signatures are listed only when more than one study has been conducted on a specific cell biological, physiological or pathological topic.
Besides bulk skeletal muscle, refined studies of muscle subtypes with an unusual histology such as extraocular muscles have been conducted [ 66 , 85 ], including the proteomic profiling of sarcomere-associated elements [ 86 ].
In addition, posttranslational changes were studied in skeletal muscle preparations by proteomics, focusing especially on protein nitration [ 87 ], carbonylation [ 88 ], glycosylation [ 89 — 91 ] and phosphorylation [ 92 , 93 ]. Comprehensive reviews have covered the critical examination of these proteomic studies [ 10 — 15 , 94 , 95 ].
Thus, instead of recapitulating the considerable impact of these early studies of muscle proteomics, this review instead outlines the more recent application of fluorescent gel electrophoresis, organelle proteomics and membrane proteomics for studying skeletal muscle tissues. Labeling of proteins with fluorescent dyes has been extensively applied in proteomic investigations [ 96 — 99 ]. However, the one technique that stands out for its potential to directly compare two different sets of protein complements is fluorescence difference in-gel electrophoresis, usually abbreviated as DIGE [ ].
This advanced gel electrophoretic method represents one of the most powerful analytical tools for conducting comparative protein biochemical investigations [ ]. The fluorescence DIGE technique covers the same type of proteins as conventional two-dimensional gel electrophoretic approaches. If proper labeling protocols are followed, the fluorescence tagging procedure does not significantly interfere with the chemical properties of proteins with respect to their gel electrophoretic mobility.
Minden et al. The DIGE technique is an ideal method for comparing entire soluble proteomes in one swift analytical approach [ ] if one accepts that proteins with extreme p I values, proteins with a very low density, certain classes of high molecular mass proteins, extremely hydrophobic proteins and elements with certain posttranslational modifications may be underrepresented in two-dimensional gel systems [ 19 , 44 — 46 ].
DIGE greatly reduces gel-to-gel variations and thereby greatly improves the evaluation of trends in changed protein expression patterns [ ]. The DIGE method has also captured a lot of attention in the field of skeletal muscle proteomics over the past few years [ 55 — 57 , 59 , 63 — 66 ].
If the experimental design of fluorescent studies maximises the sensitivity for detecting changes in protein expression levels and takes into account statistical variations in dye binding, labelling artefacts of soluble protein species can be kept to a minimum [ — ]. Therefore, reverse DIGE labelling controls are not routinely employed.
Dye-to-dye variability is usually minimal, and the findings from expression analyses with different dye combinations do not differ to a large extent. Analytical DIGE systems can be employed with two-dye or three-dye systems, depending on specific applications. Advanced DIGE, using an internal pooled standard, is a highly accurate quantitative method that enables multiple protein samples to be separated on the same two-dimensional gel. For example, a recent study conducted at our laboratory of the effects of experimental exon skipping on the dystrophic diaphragm from the x-linked muscular dystrophy mdx model of Duchenne muscular dystrophy combined the proteomic profiling of normal versus mdx versus treated mdx specimens [ 65 ].
A pool of all protein samples was also prepared and labelled with another CyDye to be employed as a standard on all gels. A pooled standard greatly aids image matching and cross-gel statistical analysis. Ideally, samples are evenly distributed between both CyDye fluors and analytical gels [ 65 ].
Introduction to proteomics: principles and applications / Nawin Mishra. p. ; cm.—(Methods of biochemical analysis ; ). Includes bibliographical references. Principles and Applications: 52 (Methods of Biochemical Analysis) Book everyone. Proteomics provides an introductory insight on proteomics, discussing the.
Figure 3 outlines the use of differential dye labelling for the analysis of two different muscle specimens. To illustrate the sensitivity of the fluorescent method, an enlarged DIGE image of distinct changes in the isoform expression pattern of myosin light chain during fast-to-slow muscle transitions is shown. Our proteomic profiling of fast muscle following chronic low-frequency stimulation has been studied by using DIGE analysis, and it clearly revealed a switch to slower isoforms of myosin light and heavy chains, as well as increased levels of oxidative enzymes [ 63 ].
Overview of the fluorescence difference in-gel electrophoretic DIGE method. Shown is a diagram of the differential labeling of muscle specimens with the fluorescent CyDyes Cy2, Cy3 and Cy5, as well as an example of a DIGE analysis of myosin light chain isoforms during fast-to-slow transitions of electrostimulated skeletal muscle. Large-scale gel electrophoretic and liquid chromatographic methods have limitations with respect to analysing highly complex protein mixtures and protein populations with an extensive dynamic expression range.
Thus, to reduce sample complexity, organelle proteomic studies have been initiated that focus on the protein complement of distinct subcellular fractions.
Organelle proteomic approaches include sample prefractionation and the use of narrow pH ranges for the isoelectric focusing of low copy number proteins in two-dimensional gels as well as in one-dimensional gels for studying hydrophobic and high molecular mass proteins [ — ]. Reference maps of subcellular fractions from skeletal muscle include microsomes, sarcolemma, cytosol, contractile apparatus and mitochondria [ — ].
Since mitochondria are involved in various diseases and cellular aging, many subcellular proteomic studies have focused on this crucial organelle [ — ]. Proteomic profiling of the mitochondria-enriched fraction from senescent rat muscle has revealed a shift to more aerobic oxidative metabolism in a slower-twitching fibre population during age-related muscle degeneration [ ]. This organelle proteomic study has identified many new potential biomarkers of sarcopenia of old age [ 14 ].
Maughan et al.
Assessing the mitochondrial membrane potential in cells and in vivo using targeted click chemistry and mass spectrometry. Skip to main content. The half-lives of other proteins of similar mass, such as apolipoproteins A-I and A-II and retinol-binding protein, are extended severalfold by binding to larger carriers. Application of bioaffinity mass spectrometry for analysis of ligands. Only a few peptides were derived from less abundant proteins such as C4, clusterin also known as apolipoprotein J , high—molecular-mass kininogen, and coagulation factor XIII.
This makes the key metabolic proteins that mediate the core glycolytic pathway ideal candidates to be studied by using mass spectrometry-based proteomics [ 95 ]. In contrast, organelle-associated proteins are usually more difficult to identify and characterise. In this respect, Figure 4 shows a novel approach for studying subcellular fractions by on-membrane digestion of electrophoretically transferred proteins.
On-membrane digestion method for muscle proteomics. The flowchart outlines the application of on-membrane digestion for the mass spectrometric identification of membrane proteins and high molecular mass proteins from skeletal muscle tissues. An inefficient trypsination of certain target proteins often hampers in-gel digestion procedures. To address this technical problem in the proteomic identification of proteins, on-membrane digestion has been developed [ — ]. The advantage of on-membrane digestion is superior protein sequence coverage [ ].
In addition, on-membrane digestion is more efficient and faster compared with conventional in-gel digestion methods [ ]. This technical fact reduces complications due to trypsin autolysis and makes the on-membrane digestion technique especially suitable for the mass spectrometric identification of low copy number proteins and large hydrophobic proteins. This technique has recently been applied to the biochemical analysis of the large-membrane cytoskeletal protein dystrophin from rabbit skeletal muscle [ ]. Standard two-dimensional gels are usually unable to separate muscle proteins with a molecular mass greater than kDa [ 56 ].