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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:
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

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