1. Introduction
Efficient protein separation is a prerequisite for a variety of bioanalytical applications [
1]. Physicochemical parameters such as size, charge and solubility of individual polypeptides have been extensively exploited to develop sophisticated techniques for the isolation of specific protein species. Electro-focusing methods and one-dimensional gel electrophoresis (GE) are long established methods of protein biochemistry. Conventional isoelectric focusing (IEF) separates proteins by differences in their isoelectric point (p
I) whereby a pH gradient along the length of a gel provides the support system for protein migration until they reach their p
I-value with no net charge [
2]. In contrast, one-dimensional gel electrophoresis in the presence of an anionic detergent, such as sodium dodecyl sulfate (SDS), is based on the separation of denatured and structurally linearized polypeptides within complex protein mixtures. Electrophoretic mobility patterns mostly depend on size differences of the denatured molecules due to the introduction of an overall negative charge [
3,
4,
5]. However, since many protein species exhibit a similar net charge or relative molecular mass, one-dimensional gel bands are often heterogeneous in composition. Once it became clear that biochemical techniques focusing on only one parameter have relatively limited separation capacity, alternative approaches were attempted. Especially the sequential usage of two independent methods promised the separation of proteins at higher resolution. The technical realization of this ground-breaking concept was the beginning of a new era in protein biochemistry. The combined property of the p
I-value of a protein and its molecular size following denaturation was successfully exploited in the development of standardized two-dimensional gel electrophoresis (2D-GE). Historical and technical aspects of combined gel electrophoretic approaches have been extensively reviewed [
6,
7,
8].
Patrick H. O’Farrell’s work set the scene for high-resolution gel electrophoresis [
9]. Many other laboratories developed similar approaches or modified the original gel electrophoretic method to adapt this technique to other analytical applications [
10,
11,
12,
13]. Both, protein biochemistry and the more recently established field of mass spectrometry (MS)-based proteomics have heavily depended on the 2D-GE method in the past, making this method one of the most commonly employed standard technique of protein separation. In addition, gel-free approaches and the usage of 1D-GE systems are frequently used for the proteomic analysis of complex tissues. The most crucial capabilities of the 2D-GE technique are the simultaneous resolution of thousands of distinct protein species within the same gel system and the reliable determination of their relative molecular mass and p
I-value, as well as their relative quantity. Especially the application of the 2D-GE method for the efficient separation of different protein isoforms with dynamic post-translational modifications (PTM) has made outstanding contributions in analytical biochemistry. In contrast to other large-scale separation approaches such as liquid chromatography (LC), individual protein spots are visualized in 2D gels so that their status in relation to fragmentation and modification can be directly accessed. Furthermore, gel-separated and -embedded proteins are relatively stable and can be safely stored for long periods of time prior to further analysis [
14,
15,
16,
17].
This article focuses on the application of the 2D-GE method in basic myology and discusses the enormous scientific impact of this method on recent proteomic studies of normal and physiologically challenged skeletal muscle tissues. This includes the systematic cataloguing of the protein constituents of different contractile fiber types and the findings from surveys of proteome-wide changes during physiological adaptations. Comparative proteomics has also played a key role in the pathobiochemical evaluation of global changes in diseased skeletal muscles [
18]. However, this topic is not addressed in this article on the biochemistry of normal, adapting and aging skeletal muscles. Several extensive reviews on the pathoproteomics of neuromuscular disorders have outlined the various proteomic techniques used in the determination of molecular and cellular mechanisms that underlie common muscle diseases [
19,
20,
21,
22]. The critical examination of how MS-based proteomics can be used for the systematic identification and biochemical characterization of novel protein biomarkers, which may be exploitable for the future design of improved predictive, diagnostic, prognostic and/or therapy-monitoring assays, has also recently been reviewed [
23]. Below sections give an overview of major proteomic studies that have employed 2D-GE methods and analyzed fiber type specification and protein changes during muscle development, fiber type transformation, exercise-induced adaptations, hypoxia-associated alterations, disuse-related muscular atrophy and skeletal muscle aging.
2. Two-Dimensional Gel Electrophoresis of Skeletal Muscle Proteins
The 2D-GE technique is a frequently used and highly reliable bioanalytical method for the systematic assessment of skeletal muscle tissues. Following tissue homogenization under optimized conditions and in the presence of a suitable protease inhibitor cocktail, a large portion of skeletal muscle proteins can be extracted without major complications due to proteolytic degradation and then separated by 2D-GE. The application of the original O’Farrell method [
9] or modified and optimized gel electrophoretic techniques [
24] played an essential role in the systematic identification and thorough characterization of the protein components that form the functional basis of skeletal muscle contractility. The PubMed databank of the US National Library of Medicine contains nearly 33,000 entries with the keyword “two-dimensional gel electrophoresis” of which over 600 publications are in relation to the combined key words “two-dimensional gel electrophoresis” and “skeletal muscle”, including from the year 2001 onwards nearly 200 papers using MS-based proteomics [
25].
Figure 1 summarizes in a histogram the number of published papers per year on this topic since 1976 and shows an increase in the usage of the 2D-GE method after the incorporation of MS-based proteomics for the routine analysis of skeletal muscle tissues since 2004. Over the last 4 decades, the combined usage of IEF in the first dimension and SDS-PAGE in the second dimension has been successfully employed to identify and characterize several thousand muscle-associated or muscle-derived protein species. These muscle 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, extracellular matrix proteins and myokines.
As outlined in
Figure 2, conventional biochemical approaches and more recently established MS-based proteomic methods have integrated the 2D-IEF/SDS-PAGE technique as a highly suitable method for detailed investigations into skeletal muscle proteins. It is important to mention that skeletal muscle tissues are heterogeneous in their composition and highly dynamic in their response to cellular, metabolic or physiological challenges [
26]. The main contractile units of an individual skeletal muscle are presented by diverse fiber populations, consisting usually of slow-oxidative, intermediate fast-glycolytic/oxidative and fast-glycolytic cell types, as well as mixed hybrid fibers. This relates to the histochemically well-defined fiber types I, IIa, IIx and IIb, and the hybrid fibers I/IIa, IIa/IIx and IIx/IIb [
27]. The internationally agreed nomenclature of muscle fiber types is based on the distribution of myosin heavy chain (MyHC) isoforms [
28]. Besides contractile fibers, muscle tissue contains motor neurons with their extensive myelin sheets, an elaborate network of capillaries, satellite cells and multiple layers of connective tissue, including the epimysium, perimysium and endomysium [
29]. Biochemical studies using homogenized tissue preparations have to take into account this cellular heterogeneity of skeletal muscles [
21], as well as the presence of actively secreted and passively released fiber-derived proteins that constitute the muscle secretome [
30].
Prior to the publication of the original O’Farrell method [
9], 2D-GE studies on skeletal muscle employed SDS-PAGE in both dimensions with differing gel concentrations. These early attempts could only separate a few distinct protein spots from muscle ribosomes [
31] and the microsomal fraction from skeletal muscles [
32]. However, following the publication of high-resolution 2D-IEF/SDS-PAGE methods [
9,
10,
11,
12], this new and more refined approach was quickly adapted in the field of basic and applied myology [
24]. Initial studies included the analysis of major structural and regulatory proteins of muscle fibers [
33,
34], the evaluation of human muscle biopsy specimens [
35,
36] and the identification of contractile protein isoforms in single skeletal muscle fibers [
37,
38]. In the pre-proteomic era of 2D-GE biochemistry [
39], the technique was extensively applied to the detailed analysis of subunit structures and isoform expression patterns of major skeletal muscle proteins and their changes during development, fiber adaptations, contractile fatigue and denervation, as reviewed by Bárány et al. [
40]. From 1995 onwards, gel-based surveys and 2D-GE databases became an integral part of skeletal muscle proteomics [
41]. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations, muscle regeneration and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Below sections review the application of the 2D-IEF/SDS-PAGE method in modern proteomics and its modifications for comparative studies using fluorescent dyes. Included are descriptions of the systematic cataloging of the assessable skeletal muscle proteome, and the comparative proteomic profiling of muscle plasticity in relation to neuromuscular activity versus disuse atrophy.
3. Cataloguing of the Skeletal Muscle Proteome Using Two-Dimensional Gel Electrophoresis
MS-based muscle proteomics was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation [
21,
42,
43]. General technical aspects of the most frequently employed 2D-GE methods in the proteomic profiling of crude tissue extracts, subcellular fractions or isolated protein complexes have been extensively discussed and reviewed [
15,
17,
44,
45,
46]. Over the last few years, several excellent methods books have been published that focus on specific aspects of the many modifications used in routine 2D-GE and proteome analysis protocols. These comprehensive collections of detailed method descriptions have been edited by experts in the field, including Link [
47], Reinders and Sickmann [
48], Cramer and Westermeier [
49], Kurien and Scofield [
50], and Marengo and Robotti [
51]. In-gel staining methods and routinely used detection technologies for studying 2D protein spot patterns have also been extensively described and critically examined in numerous publications [
52,
53,
54,
55,
56,
57,
58,
59,
60]. The rapidly moving field of MS methodology analyzing peptides obtained from the proteolytic digestion of proteins is the subject of many excellent articles that outline in detail the many instruments and approaches available in modern proteomics research [
61,
62,
63,
64]. These optimized gel-based protein separation approaches, highly reliable pre-electrophoretic or in-gel staining techniques and sophisticated MS methods for the unequivocal identification of individual protein species have been extensively used in skeletal muscle proteomics [
20,
21,
22]. In relation to skeletal muscle biochemistry and proteomics, detailed step-by-step descriptions of tissue sample preparation, protein extraction, protein solubilization, IEF, SDS-PAGE, pre-electrophoretic protein labeling, post-electrophoretic protein staining and subsequent MS analysis of proteins of interest following controlled proteolytic degradation have been published in comprehensive methods papers [
65,
66,
67,
68].
In the first volume of the journal
Proteomics, launched in 2001, publications by Hochstrasser and colleagues [
69] and Dunn and co-workers [
70] set the scene for 2D gel-based skeletal muscle proteomics. Their initial studies identified over 70 proteins each from normal mouse and rat skeletal muscle homogenates, including many sarcoplasmic, metabolic and myofibrillary proteins such as various myosin subunits, actin isoforms, regulatory sarcomeric proteins, glycolytic enzymes, mitochondrial proteins and molecular chaperones [
69,
70]. The application of the 2D-GE method in combination with MS technology for cataloging mouse skeletal muscle was part of extending the SWISS-2DPAGE database to include proteomic maps of major types of tissue [
69]. A variety of databases exist for the image comparison of 2D gels, the identification of internet-based gel images, the cataloguing of results from systematic 2D-GE analyses and the integration of electrophoretic and mass spectral data from proteomic analyses, including World-2DPAGE Constellation SWISS-2DPAGE, WEB P.A.G.E, Flicker, GELBANK, Open2Dprot Project, LECB 2-D PAGE Gel Images Data Sets, 2DWG, ProteomeWeb, PHProteomicsDB and pProRep [
71,
72,
73,
74,
75,
76,
77,
78]. In 2001, the new field of skeletal muscle proteomics was further developed by the systematic identification of sarcoplasmic proteins from several hake species [
79] and the mass spectrometric characterization of bovine myosin light chain MLC1f polymorphism following 2D-GE separation [
80]. Potential technical shortcomings of 2D-GE for the comprehensive separation of highly complex protein mixtures have often been discussed in the past and compared to the perceived superiority of LC-based methods [
17,
81,
82]. In our opinion, both protein separation techniques should be seen as complementary proteomic methods and be used in combination to achieve the maximum coverage of the assessable proteome of a particular biological specimen.
In the case of skeletal muscle fibers, approximately half of the protein constituents belong to the sarcomere units that are made up of large numbers of isoforms of myosin heavy chains (MyHC), myosin light chains (MLC), actins (ACT), troponins (TN) and tropomyosins (TM). These contractile and regulatory protein species are routinely identified by gel-based proteomics [
43], demonstrating the usefulness of 2D-GE for the classification of muscle types and fiber specification. Metabolic enzymes are also highly abundant in muscle tissues and straightforwardly assessable by gel-based proteomics [
83]. Prior to outlining the many advantages and bioanalytical applications of 2D-GE in skeletal muscle proteomics in subsequent sections, the below listing highlights certain issues that may hamper gel-based methods and other types of analyses. The most frequently encountered biological and technical complications (and some alternative approaches to avoid these potentially limiting factors) are:
Possible under-estimation of particular types of proteins, including highly hydrophobic proteins, very high-molecular-mass proteins and low-copy-number proteins. Changing gel conditions, the introduction of suitable pre- and post-fractionation steps, as well as higher sensitivity detection protocols can often overcome some of these technical limitations [
84,
85,
86,
87].
Hypothetical under-representation or 2D streaking of proteins with extreme p
I-values, which however depends heavily on the particular IEF conditions employed in the first dimensional separation step. Often very acidic protein species form vertical streaking patterns at the pH 3 region and very basic proteins at the pH 11 region. To at least partially overcome this problem, the usage of narrow-range immobilized pH gradients can be applied for zooming in on protein species that do not fall into the commonly applied range of approximately p
I 3 to 11 [
88,
89,
90,
91]. In addition, combining the findings from several different IEF gels in the first dimension with slightly overlapping p
I-values can be advantageous for producing more comprehensive protein coverage [
15,
92,
93].
Potentially restricted separation of complex protein mixtures with greatly differing molecular masses using routine 2D-GE approaches. Often the usage of large-scale gels, optimized gradient SDS-PAGE slab gel systems in the second dimension and the reduction of sample complexity can overcome some of these technical problems and be used to cover protein species that do not fall into in the routinely analyzed range of approximately 10 to 250 kDa [
67,
94].
Latent cross-contamination of individual 2D protein spots through highly abundant polypeptides that are dragged throughout the 2D gel system due to their exceedingly high density. These abnormal electrophoretic mobility patterns of particular proteins cause a certain degree of 2D streaking, which can be minimized by (i) decreasing the total amount of protein loading; (ii) using very large gel systems with a higher discriminatory capacity and/or (iii) applying optimized pre-fractionation techniques to decisively decrease sample complexity [
95,
96,
97,
98]. Artifacts can be kept to a minimum using 50 to 200 μg of total protein in first dimension gels. Lower protein concentrations usually result in weak staining patterns. Comparative studies with fluorescent dyes give optimum results with approximately 50 μg of protein per sample.
Potential discrepancies between the findings from the densitometric scanning of gel images and the MS-based protein identification in case of a heterogeneous composition of a single 2D protein spot. For example, if a protein spot contains more than one protein species and the most abundant protein is not as susceptible to digestion as the low-copy-number proteins in its vicinity, then the concentration change of this 2D protein spot (as determined by densitometric scanning) may be misleading. However, this analytical complication is a relatively rare occurrence and the use of simple post-fractionation approaches and/or independent verification of gel-based proteomic data by immunoblotting surveys or immunofluorescence microscopical analysis can effectively assess the rate of these kinds of analytical discrepancies [
21].
In relation to the analysis of skeletal muscle preparations by 2D-GE, the above listed technical and biological issues may complicate especially the routine analysis of the many very large proteins present in the neuromuscular system, such as titin (3700 kDa), nebulin (800 kDa), obscurin (720 kDa), the ryanodine receptor Ca
2+-release channel (565 kDa) and dystrophin (427 kDa). 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. Depending on the overall loading capacity of a particular 2D gel, the high density of some isoforms of these sarcomeric proteins can cause a select amount of cross-contamination in particular regions of a 2D-GE system [
67,
103].
Despite these bioanalytical limitations, the many technical advantages of the 2D-GE approach far outweigh the potential shortcomings of this extensively used protein separation method, as listed below:
Extremely reliable protein separation system that can be routinely used in large-scale and high-throughput proteomic surveys. Multi-gel systems using large buffer tanks can run a considerable number of 2D gels in parallel making this approach both cost-effective and highly reproducible for systematic biochemical studies [
15,
16,
17].
Staining of 2D gels with highly sensitive dyes ranging from colloidal Coomassie Blue to silver stains to a variety of fluorescent dyes can visualize a wide dynamic range of proteins of differing abundance [
56,
57,
58,
104].
Technical provision of a bioanalytical platform that is ideally suited for the subsequent identification of specific protein isoforms and their PTMs [
46,
47,
48]. Many in-gel staining or labeling methods can specifically highlight PTMs, such as enzyme-conjugated lectin labeling or Pro-Q Emerald staining for glycosylation or the fluorescent Pro-Q Diamond dye for phosphorylation [
58,
105,
106,
107].
Direct visualization of proteins of interest as discrete 2D spots, enabling the exact evaluation of the characteristic combination of the p
I-value and relative molecular mass of a particular protein subunit or isoform. This provides a unique analytical advantage over simpler 1D gel systems that display heterogeneous protein bands or LC methods. Often MS data from LC-based analyses do not given efficient information on sequence coverage to unequivocally determine whether a fully intact protein species or fragments have been detected. In contrast, proteomic data from the analysis of distinct 2D-GE spots can be directly correlated with the electrophoretic mobility and thereby the relative molecular mass of the protein of interest [
21].
Since potential discrepancies between the mass spectrometric identification of a protein and its position in a 2D gel in relation to its p
I-value and/or molecular mass can be easily assessed, the rate of false positive protein hits can be conveniently measured and swiftly eliminated from the final list of altered protein species. Additional analyses can then determine whether an abnormal or unexpected electrophoretic mobility pattern is due to protein degradation, protein clustering or a technical artifact caused by 2D streaking and cross-contamination [
18].
Rapid and quantitative analyses of paired protein samples can be conducted. An example of an extremely powerful comparative 2D-GE method is the fluorescence 2D-DIGE technique [
108] that eliminates gel-to-gel variations by the differential pre-electrophoretic labeling of protein fractions and the subsequent separation on the same 2D gel followed by image analysis [
109]. See below section for details on the DIGE method and its application in skeletal muscle proteomics.
Since the beginning of the new millennium, several thousand muscle-associated or muscle-derived protein species and subgroups with particular PTMs have been identified and used to establish the skeletal muscle proteome. A large proportion of the comprehensive cataloguing of total muscle tissue extracts from various species and subtypes of muscle was carried out by 2D-IEF/SDS-PAGE [
69,
70,
110,
111,
112,
113,
114,
115,
116,
117,
118,
119,
120,
121,
122,
123,
124,
125,
126]. Subcellular fractions of skeletal muscles have also been studied by 2D-GE and MS analysis [
127], including nuclei [
128], mitochondria [
129,
130,
131,
132], the contractile apparatus [
133], cytosol [
128] and the muscle secretome [
134,
135]. In addition, 1D-GE and on-membrane digestion has been used to characterize the sarcolemma [
99] and sarcoplasmic reticulum [
101] from skeletal muscle preparations. Major classes of PTMs, such as muscle protein nitration, glycosylation and phosphorylation were determined by 2D-GE methodologies [
105,
106,
136,
137]. These gel-based proteomic cataloguing studies were supplemented with data from alternative GE methods and a large number of LC-based proteomic investigations [
138,
139,
140,
141,
142,
143,
144,
145,
146,
147,
148,
149,
150] to fully comprehend the enormous complexity of the muscle proteome [
21]. Details of major 2D gel-based studies for the establishment of the skeletal muscle proteome are listed in
Table 1. Within this large cohort of skeletal muscle proteins, fiber type-specific expression patterns of a few hundred muscle proteins have been established by 2D-GE analysis, confirming the molecular and cellular heterogeneity between predominantly fast-twitching and slow-twitching muscles [
113,
114,
115,
116,
117,
118]. This important topic of skeletal muscle physiology and the major changes that occur during fiber transitions is discussed in the below section on comparative proteomics.
5. Conclusions
Over the last 40 years, protein separation has been carried out by sophisticated 2D-GE techniques and established this method as a highly suitable and versatile approach for the systematic analysis and characterization of the skeletal muscle proteome. In basic and applied myology, the application of the original O’Farrell method or slightly modified versions has resulted in the cataloging of several thousand distinct muscle protein isoforms and the identification of hundreds of fiber type-specific protein species. Gel-based proteomic studies have established a variety of protein changes in physiologically challenged skeletal muscles, including the effects of myogenesis, exercise, regeneration, hypoxia, prolonged disuse and natural aging. In this regard, the 2D-GE method has played an essential role in modern muscle biology and the systematic identification of the molecular components that form the functional units of contractility and adaptability to changed functional demands. The extensive usage of 2D-GE has been instrumental in the establishment of the highly dynamic skeletal muscle proteome signature that is characterized by an extremely diverse population of protein species. Crucial issues in muscle tissue proteomics are optimum protein extraction and efficient protein separation prior to MS analysis. Currently no single protein biochemical method is capable of separating all of the molecular species that constitute the skeletal muscle proteome. The considerable differences in charge, size, solubility and abundance may result in the under-representation of specific subtypes of peptides and proteins. Independent of the specific separation approach, such as gel-based techniques or liquid chromatography, it is currently not possible to cover the entire range of muscle protein isoforms in large-scale and high-throughput analyses. Technical limitations of the 2D-GE method may especially affect the proteomic analysis of very high-molecular-mass proteins, integral membrane proteins and low-abundance proteins. Although liquid chromatography has the advantage of being able to efficiently separate highly hydrophobic proteins, this method cannot routinely determine the characteristic combination of the relative molecular mass and the pI-value of a specific protein of interest. Hence, the direct visualization of muscle proteins as discrete 2D spots can be useful to clearly determine whether smaller fragments or a full-length protein have been identified by proteomics. Therefore, using a combination of different biochemical techniques with overlapping separation capabilities for dissimilar subtypes of proteins would be the best way to cover the majority of muscle protein species in comprehensive proteomic studies. With the rapid advances in MS technology, it can be expected that future proteomic investigations will establish an even more refined understanding of the interactions between regulatory proteins, contractile elements, cytoskeletal proteins, extracellular matrix proteins, metabolic enzymes, signaling complexes, ion-handling complexes and molecular chaperones to form the structural and functional basis of the neuromuscular system.