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ACTN3 genotype is associated with human elite athletic performance. American Journal of Human Genetics, 73, 627–631. Yoshihara, A., Tobina, T., Yamaga, T., Ayabe, M., Yoshitake, Y., Kimura, Y., Shimada, M., Nishimuta, M., Nakagawa, N., Ohashi, M., Hanada, N., Tanaka, H., Kiyonaga, A., Miyazaki, H. (2009), Physical function is weakly associated with angiotensin-converting enzyme gene I/D polymorphism in elderly Japanese subjects. Gerontology, 55, 387–392. 257Genetic Variation and Skeletal Muscle Traits: Implications for Sarcopenia Zemel, S., Boartolomei, M.S., Tilghman, S.M. (1992). Physical linkage of two mammalian imprinted genes, H19 and insulin-like growth factor 2. Nature Genetics, 2, 61–65. Zhai, G., Stankovich, J., Ding, C., Scott, F., Cicuttini, F., Jones, G. (2004). The genetic contribu- tion to muscle strength, knee pain, cartilage volume, bone size, and radiographic osteoarthritis: a sibpair study. Arthritis and Rheumatism, 50, 805–810. Zhai, G., Ding, C., Stankovich, J., Cicuttini, F., Jones, G. (2005). The genetic contribution to longitudinal changes in knee structure and muscle strength: a sibpair study. Arthritis and Rheumatism, 52, 2830–2834. 259 G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness, DOI 10.1007/978-90-481-9713-2_12, © Springer Science+Business Media B.V. 2011 Abstract Muscle proteomics is concerned with the large-scale profiling of the protein complement from contractile tissues in order to enhance our biochemical knowledge of fundamental physiological processes, as well as the pathophysiological mechanisms that underlie neuromuscular disorders. Since the loss of skeletal muscle mass and strength is one of the most striking features of the senescent body, a large number of proteomic studies have recently attempted the global analysis of age-related fibre degeneration. Although the large size of the muscle proteome and its broad range of expression levels complicates a comprehensive cataloguing of the entire muscle pro- tein complement, mass spectrometry-based proteomic studies have succeeded in the identification of many novel sarcopenia-specific markers. Changes in the expression of affected muscle proteins, as well as altered post-translational modifications, can now be used to establish a reliable biomarker signature of age-dependent fibre wasting. Muscle proteins that are changed during aging belong to the regulatory and contractile elements of the actomyosin apparatus, key bioenergetic pathways, the myofibrillar remodeling machinery and the cellular stress response. The proteomic profiling of crude muscle extracts and distinct subcellular fractions agrees with the notion that sarcopenia of old age is due to a multi-factorial pathology. Changes in muscle markers of the contractile apparatus and energy metabolism strongly indicate a fast-to-slow fibre transition pro- cess and a shift to more aerobic-oxidative metabolism during aging. In the long-term, newly established biomarkers of sarcopenia might be useful for the design of improved diagnostic procedures and the identification of new therapeutic targets. Keywords Mass spectrometry • Muscle aging • Muscle proteome • Muscle proteomics • Sarcopenia K. O’Connell, J. Gannon, and K. Ohlendieck (*) Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland e-mail: kay.ohlendieck@nuim.ie P. Doran Department of Biological Chemistry, University of California, Los Angeles, CA, USA P. Donoghue Conway Institute, University College Dublin, Belfield, Ireland Proteomic and Biochemical Profiling of Aged Skeletal Muscle Kathleen O’Connell, Philip Doran, Joan Gannon, Pamela Donoghue, and Kay Ohlendieck 260 K. O’Connell et al. 1 Introduction Since skeletal muscle fibres represent the most abundant type of tissue in mammalians, primary pathological changes in the neuromuscular system have profound secondary effects on overall body homeostasis and bioenergetic requirements. It is therefore not surprising that patients suffering from inherited muscular dystrophies and related muscle wasting disorders have also functional impairments in other organ systems (Emery and Muntoni 2003). However, loss in skeletal muscle mass and associated contractile weakness may also occur as a critical co-morbidity in human disease. Secondary muscular dysfunction is seen in common disorders such as diabetes mellitus (Phielix and Mensink 2008), the metabolic syndrome (Wells et al. 2008), congestive heart disease (Dalla Libera et al. 2008), cancer-associated cachexia (Melstrom et al. 2007), sepsis (Smith et al. 2008), renal failure (Adams and Vaziri 2006) and chronic obstructive pulmonary disease (Wuest and Degens 2007). Importantly, during the natural aging process, a gradual reduction in muscle mass and a progressive decline in contractile strength is seen in all humans to a varying degree (Thompson 2009). It is not well understood whether muscle degen- eration during aging is primarily due to abnormalities in the contractile tissue itself or a secondary consequence of severely impaired innervation patterns (Carlson 2004). The results from a large number of cross-sectional and longitudinal studies do not agree on the exact extent of age-dependent muscle degeneration (Forbes and Reina 1970; Baumgartner et al. 1995; Lindle et al. 1997; Proctor et al. 1999; Melton et al. 2000; Janssen et al. 2002) and how individual muscles are differentially affected during aging (Frontera et al. 2008), but concur that human aging is clearly associated with a severely impaired structure and function of the cells comprising the musculoskeletal system (Vandervoort 2002). Progressive muscular dysfunction may prevent elderly patients from living an independent life and may require outside help despite the lack of other medical ailments (Rolland et al. 2008; Thompson 2009). The vastly improved availability of high-quality nutrients, enhanced hygiene, superior medical care and hugely improved pharmacological interventions have achieved an unprecedented extension of human longevity over the last few decades. It is now imperative to acquire the scientific basis of evidence to aid the development of new therapeutic strategies for the promotion of healthy aging (Lynch et al. 2007). In this respect, it is crucial to elucidate the molecular and cellular mechanisms that render the aged neuromus- cular system more susceptible to degeneration (Doherty 2003). High-throughput and large-scale approaches used in the emerging biomedical fields of genomics, proteomics and metabolomics suggest themselves as ideal tools for the identifica- tion of novel markers of sarcopenia (Doran et al. 2007a). Currently, both proper diagnostic criteria to fully describe the different stages of skeletal muscle aging and suitable treatment options to reverse sarcopenia are lacking. The establishment of a disease- and stage-specific biomarker signature of sarcopenia would therefore greatly aid in the development of better diagnostic tools and the identification of novel therapeutic targets to treat age-dependent fibre degeneration. 261Proteomic and Biochemical Profiling of Aged Skeletal Muscle 2 Skeletal Muscle Proteomics In the post-genomic era, skeletal muscle proteomics attempts the global profiling of voluntary contractile tissues in order to identify and catalogue the entire fibre protein complement and determine alterations in the abundance, post-translational modifications and oligomeric status of muscle proteins in development, differentia- tion, disease and aging (Isfort 2002). This includes the proteomic profiling of motor units, distinct muscles, individual classes of muscle fibres and defined subcellular fractions such as mitochondria, the contractile apparatus or the sarcoplasmic reticu- lum. Muscle proteomics employs standardized biochemical methodology to effi- ciently separate, unequivocally identify and comprehensively characterise muscle-associated protein species. The techniques of choice are mass spectrometric peptide fingerprinting for routine high-throughput analyses, and peptide fragmenta- tion analysis and chemical peptide sequencing for targeted proteomics (Aebersold and Mann 2003). The long-term goal of muscle proteomics is to decisively improve our biochemical knowledge of fundamental physiological processes related to the many cellular functions of contractile tissues, as well as the elucidation of the molecular mechanisms that underlie neuromuscular pathology. 2.1 Mass Spectrometry-Based Proteomics In contrast to the traditional reductionist approach focusing on specific proteins, complexes or pathways, modern proteomics attempts to carry out large-scale high- throughput analyses of entire cellular protein complements (de Hoog and Mann 2004). The combination of highly accurate mass spectrometric methods and opti- mized electrophoretic and chromatographic separation technology has provided an unprecedented capability for the swift qualitative and quantitative analysis of large numbers of proteins (Ferguson and Smith 2003). Since mass spectrometric peptide fingerprinting or peptide fragmentation techniques are dependent on the existence of suitable protein- or DNA-based databanks for sequence comparisons, the informa- tion generated by the human genome project and related sequencing projects for other species form an integral part of any proteomic workflow. Modern proteomics can identify individual protein isoforms and determine potential changes in their concentration or post-translational modifications from extremely small amounts of biological material. Especially the introduction of differential fluorescent tagging approaches has improved the simultaneous analysis of several proteomes (Viswanathan et al. 2006). Muscle proteomics in particular is concerned with the global identification, cataloguing and comparative analysis of the protein comple- ment present in distinct subcellular fibre fractions, differing muscle fibres and subtypes of muscles (Isfort 2002). Optimized biochemical methods are used for the comprehensive and reproduc- ible separation of the accessible muscle proteome or subproteomes. Subsequently 262 K. O’Connell et al. the individual constituents of mixtures of peptides, proteins and supramolecular complexes are rapidly identified and characterized by a variety of mass spectromet- ric techniques (Domon and Aebersold 2006). In muscle biology, the majority of proteomic profiling exercises have been carried out with gel electrophoretic separa- tion methods, as reviewed by Doran et al. (2007b). See the flow chart of Fig. 1 for an outline of a typical proteomic profiling exercise that employs fluorescent tagging technology. Unlu and co-workers (1997) first described this powerful comparative method and Tonge et al. (2001) have evaluated the capabilities of its 2D software analysis program. Fluorescent difference in-gel electrophoresis, usually abbrevi- ated as DIGE analysis, represents a highly accurate quantitative technique that enables the separation of multiple proteomes on the same two-dimensional gel, thereby greatly reducing the introduction of potential artifacts due to gel-to-gel variations (Marouga et al. 2005). Although all gel-based separation techniques have their limitations, two-dimensional methods with isoelectric focusing in the first dimension and ionic detergent-based slab gel electrophoresis in the second dimen- sion are still the method of choice for most proteomic pilot studies (Gorg et al. 2004; Wittmann-Liebold et al. 2006). Two-dimensional gel electrophoresis under- estimates the number of integral membrane proteins present in a crude tissue extract and does not properly separate or account for protein species with extreme pI-values, very large molecular masses, low abundance and/or extensive post- translational modifications. It is important to keep these technical restrictions in mind when analysing skeletal muscle fibres. Recently, the application of detergent extraction procedures and the careful application of subcellular fractionation pro- cedures has improved the scope of proteomic investigations and has included many integral components in subproteomic approaches (Sadowski et al. 2008; Zheng and Foster 2009). Thus, crucial proteins involved in the regulation of mitochondria, plasmalemma, endoplasmic reticulum, nucleus and cytosol are now routinely included in the subproteomic screening of normal and pathological tissue prepara- tions (Tan et al. 2008). The proteomic identification of proteins of interest is usually accomplished by standardized biochemical techniques, such as mass spectrometric peptide finger- printing, peptide fragmentation analysis, chemical peptide sequencing, the com- parison of the relative electrophoretic mobility using two-dimensional gel databanks, immunoblotting surveys employing monoclonal antibody libraries and large-scale microscopical screening. The core technique of most proteomic studies is repre- sented by mass spectrometry whereby a variety of instruments are commonly employed for the identification and characterization of biomolecules. Mass spec- trometers produce and separate ions according to their mass-to-charge ratio (m/z). The suitability of mass spectrometric instruments is defined by their resolving power, i.e. the analytical ability to differentiate between two ions of similar mass, and most importantly by their mass accuracy (Domon and Aebersold 2006). Electromagnetic fields are used to separate ions derived from biomolecules under vacuum conditions. Mass spectrometers consist of a sample introduction device, an ionization source, a mass analyzer, a detector and a digitizer. Hence, the core func- tions of these components are ion generation, ion separation, ion detection and the 263Proteomic and Biochemical Profiling of Aged Skeletal Muscle recording of a mass spectrum (Canas et al. 2006). The development of two key methods, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionisation (ESI), has improved the large-scale analysis of complex protein mixtures to an unprecedented extent (Fenn et al. 1989; Zaluzec et al. 1995). These mass spectrometric techniques can therefore be considered the key facilitators of protein biochemistry that have actually enabled the establishment of modern proteomics. Mass spectrometric peptide fingerprinting relies on the assumption that the con- trolled digestion of a protein results in the generation of a unique set of peptides that exhibit a highly reproducible combination of molecular masses (Webster and Oxley 2005). The comparison of the determined molecular masses of a sub-set of Fig. 1 Overview of proteomic difference in-gel electrophoretic analysis. Shown is the routine proteomic workflow employed for the standardized identification of novel protein biomarkers. The constituents of proteomes or subproteomes are fluorescently tagged and then separated by two-dimensional gel electrophoresis, using isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Following fluorescent difference in-gel electrophoresis (DIGE), proteins are identified by matrix- assisted laser desorption/ionization time-of-flight (MALDI-ToF) or electrospray ionisation (ESI) mass spectrometry (MS) 264 K. O’Connell et al. trypsin-generated peptides with theoretical in silico generated peptide masses leads to the identification of a specific protein species. A certain degree of proteolytic miscleavage has to be taking into account during the bioinfornatic analysis. In the case of muscle proteins, the exhaustive digestion with sequencing-grade trypsin usually produces a distinct peptide population ranging in molecular mass from approximately 500–2,500 kDa (Doran et al. 2007b). MALDI-based Time-of-Flight (ToF) mass spectrometry involves the irradiation of a co-precipitate, consisting of trypsin-generated peptides and a suitable UV-light absorbing matrix, by a nano- second laser pulse. Since different ions traverse a constant electric field according to their mass-to-charge ratio, a differential signal is generated for individual ions when they reach the detector, which transforms analogue signals into digital signals and records a mass spectrum. MALDI-ToF mass spectrometry is an extremely robust, rapid and cost-effective system for the high-throughput identification of unknown proteins (Webster and Oxley 2005). However, for targeted proteomics and the generation of large data sets of peptide sequences and the evaluation of post- translational modifications, ESI is the preferred method of choice. The ESI tech- nique is based on the fact that high voltage triggers an electric spray in a liquid flowing through a narrow capillary. Charged small droplets are formed in a solution of peptides and suspended in a gaseous atmosphere. During an evaporation process, charged peptide analytes escape from micro-drops and are then analyzed by mass spectrometry (Fenn et al. 1989). See Table 1 for an example of the proteomic iden- tification of typical muscle biomarkers. Shown are the primary sequences of pep- tides generated from mitochondrial ATP synthase and pyruvate dehydrogenase from aged skeletal muscle using ESI-MS/MS technology. The application of ESI- and MALDI-based methodology for studying complex mixtures of biomolecules has revolutionized biochemical research. With respect to muscle biology, the appli- cation of state-of-the-art genomic, proteomic and metabolomic approaches has at least partially overcome the problems associated with the traditional reductionist approach investigating individual genes or single proteins. In the future, it is hoped that high-throughput methodology will enable a detailed molecular understanding of biological problems at the systems level (Aggarwal and Lee 2003), including sarcopenia of old age. 2.2 Proteomic Profiling of Skeletal Muscle A motor unit consists of a single a-motor neuron and all its innervated contractile fibres (Chan et al. 2001). The hierarchy of biological organization within a func- tional motor unit is represented in ascending order by the genome of the nerve and its corresponding muscle fibres, their transcriptomes, subproteomes and lastly the total neuromuscular proteome (Doran et al. 2007b). Although a recent study on muscle aging has attempted the simultaneous proteomic profiling of both rat sciatic nerve and gastrocnemius muscle (Capitanio et al. 2009), most proteomic studies on skeletal muscle have focused on the fibre population without its neuronal elements 265Proteomic and Biochemical Profiling of Aged Skeletal Muscle Table 1 Proteomic identification of mitochondrial markers in aged rat skeletal muscle using ESI-MS/MS technology Name of protein Peptide sequence Accession no. Isolectric point (pI) Molecular mass (kDa) Peptides matched Mascot score % coverage Mitochondrial ATP Synthase D Chain KAIGNALKS ATP5H_RAT 6.2 18.7 13 598 86 KIPVPEDKY KYTALVDAEEKE KSWNETFHTRL KNCAQFVTGSQARV KYNALKIPVPEDKY KYTALVDAEEKEDVKN RANVDKPGLVDDFKNKY RKYPYWPHQPIENL KTIDWVSFVEIMPQNQKA RLASLSEKPPAIDWAYYRA KNMIPFDQMTIDDLNEVFPETKL KIKNMIPFDQMTIDDLNEVFPETKL Pyruvate dehydrogenase KDIIFAIKK Q6AY95_RAT 6.2 39.3 15 584 24 KDFLIPIGKA KDIIFAIKKT KVVSPWNSEDAKG RVTGADVPMPYAKI KILEDNSIPQVKD RVTGADVPMPYAKI KEGIECEVINLRT REAINQGMDEELERD RIMEGPAFNFLDAPAVRV KVFLLGEEVAQYDGAYKV RTIRPMDIEAIEASVMKT KTYYMSAGLQPVPIVFRG REAINQGMDEELERDEKV KSAIRDDNPVVMLENELMYGVAFELPTEAQSKD . changes in knee structure and muscle strength: a sibpair study. Arthritis and Rheumatism, 52, 2830–2834. 259 G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness, DOI 10.1007/978-90-481-9713-2_12,. sarcopenia might be useful for the design of improved diagnostic procedures and the identification of new therapeutic targets. Keywords Mass spectrometry • Muscle aging • Muscle proteome • Muscle. failure (Adams and Vaziri 2006) and chronic obstructive pulmonary disease (Wuest and Degens 2007). Importantly, during the natural aging process, a gradual reduction in muscle mass and a progressive