Pamela Donoghue

Proteomics of skeletal muscle aging

Extended human longevity has resulted in increasing numbers of elderly persons in the general population. However, old age is also associated with a variety of serious physical disorders. Frailty among sedentary elderly patients is related to the impaired structure and function of contractile fibers. Biochemical research into cellular mechanisms that underlie sarcopenia promises to acquire the scientific basis of evidence to aid the development of new diagnostic and therapeutic strategies. The recent application of MS‐based proteomic methodology has identified a large cohort of disease‐specific markers of sarcopenia. This review critically examines the biomedical implications of the results obtained from the proteomic screening of both aged human muscle and established animal models of sarcopenia. Substantial alterations in proteins involved in key metabolic pathways, regulatory and contractile elements of the actomyosin apparatus, myofibrillar remodeling and the cellular stress response are discussed. A multi‐factorial etiology appears to be the basis for a slower‐twitching aged fiber population, which exhibits a shift to more aerobic‐oxidative metabolism. It is hoped that the detailed biomedical characterization of the newly identified biomarkers of sarcopenia will translate into better treatment options for reversing age‐dependent muscle degeneration, which could improve the standard of living for a large portion of society.

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue.

Contractile weakness and loss of muscle mass are critical features of the aging process in mammalians. Age-related fibre wasting has a profound effect on muscle metabolism, fibre type distribution and the overall physiological integrity of the neuromuscular system. This study has used mass spectrometry-based proteomics to investigate the fate of the aging rat muscle proteome. Using nonionic detergent phase extraction, this report shows that the aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in both the detergent-extracted fraction and the aqueous protein complement from senescent muscle tissue. In the detergent-extracted fraction, the expression of ATP synthase, isocitrate dehydrogenase, enolase, tropomyosin and beta-actin was increased. Different isoforms of creatine kinase and prohibitin showed differential changes. In the aqueous fraction, malate dehydrogenase, sulfotransferase, triosephosphate isomerase, aldolase, cofilin-2 and lactate dehydrogenase showed increased levels. Interestingly, differential effects on dissimilar 2-D spots of the same protein species were shown for Cu/Zn superoxide dismutase, albumin, annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The majority of altered proteins were found to be involved in mitochondrial metabolism, glycolysis, metabolic transportation, regulatory processes, the cellular stress response, detoxification mechanisms and muscle contraction.

Nonionic detergent phase extraction for the proteomic analysis of heart membrane proteins using label-free LC-MS.

Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in the Western world and can result from either systemic disease (e.g., hypertensive heart disease, ischemic heart disease) or specific heart muscle disease (e.g., dilated cardiomyopathy/DCM). Subproteome analysis of such disease subsets affords a reduction in sample complexity, potentially revealing biomarkers of cardiac failure that would otherwise remain undiscovered in proteome wide studies. Label‐free nanoscale LC‐MS has been applied in this study to validate a Triton X‐114‐based phase enrichment method for cardiac membrane proteins. Annotation of the subcellular location combined with GRAVY score analysis indicates a clear separation between soluble and membrane‐bound proteins with an enrichment of over 62% for this protein subset. LC‐MS allowed confident identification and annotation of hydrophobic proteins in this control sample pilot study and demonstrates the power of the proposed technique to extract integral membrane‐bound proteins. This approach should be applicable to a wider scale study of disease‐associated changes in the cardiac membrane subproteome.

CyDye immunoblotting for proteomics: co-detection of specific immunoreactive and total protein profiles.

The development of ECL‐Plex CyDye‐conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre‐labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2‐D immunoprobing and protein confirmation following MS analysis.

A fluorescent codetection system for immunoblotting and proteomics through ECL-Plex and CyDye labeling.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis has been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with the recently developed ECL-Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27-kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

BSPR/EBI 2007 meeting report – Integrative Proteomics: From Molecules to Systems July 25–27, 2007 Wellcome Trust Conference Centre, Hinxton, UK

This report reviews the joint British Society for Proteome Research (BSPR) and European Bioinformatics Institute (EBI) 2007 meeting, ‘Integrative Proteomics: From Molecules to Systems’ which took place at the Wellcome Trust Conference Centre, Hinxton, UK, from 25th to 27th July. The aim of this years meeting was to explore how the integration of ‘omic’ technologies can lead to a comprehensive understanding of cellular organization, differentiation and signalling. Studies investigating protein–protein interactions and trafficking illustrated how the combination of proteomics and bioinformatics is allowing systems biology to develop as a discipline in its own right.

Proteomic and Biochemical Profiling of Aged Skeletal Muscle

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 protein 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 process 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.