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.

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.

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.

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.

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.

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 year's 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.

Characterization of the phospholemman knockout mouse heart: depressed left ventricular function with increased Na-K-ATPase activity.

Phospholemman (PLM, FXYD1), abundantly expressed in the heart, is the primary cardiac sarcolemmal substrate for PKA and PKC. Evidence supports the hypothesis that PLM is part of the cardiac Na-K pump complex and provides the link between kinase activity and pump modulation. PLM has also been proposed to modulate Na/Ca exchanger activity and may be involved in cell volume regulation. This study characterized the phenotype of the PLM knockout (KO) mouse heart to further our understanding of PLM function in the heart. PLM KO mice were bred on a congenic C57/BL6 background. In vivo conductance catheter measurements exhibited a mildly depressed cardiac contractile function in PLM KO mice, which was exacerbated when hearts were isolated and Langendorff perfused. There were no significant differences in action potential morphology in paced Langendorff-perfused hearts. Depressed contractile function was associated with a mild cardiac hypertrophy in PLM KO mice. Biochemical analysis of crude ventricular homogenates showed a significant increase in Na-K-ATPase activity in PLM KO hearts compared with wild-type controls. SDS-PAGE and Western blot analysis of ventricular homogenates revealed small, nonsignificant changes in Na- K-ATPase subunit expression, with two-dimensional gel (isoelectric focusing, SDS-PAGE) analysis revealing minimal changes in ventricular protein expression, indicating that deletion of PLM was the primary reason for the observed PLM KO phenotype. These studies demonstrate that PLM plays an important role in the contractile function of the normoxic mouse heart. Data are consistent with the hypothesis that PLM modulates Na-K-ATPase activity, indirectly affecting intracellular Ca and hence contractile function.

Proteomic profiling of chronic low-frequency stimulated fast muscle.

Skeletal muscle fibre transitions occur in many biological processes, in response to alterations in neuromuscular activity, in muscular disorders, during age-induced muscle wasting and in myogenesis. It was therefore of interest to perform a comprehensive proteomic profiling of muscle transformation. Chronic low-frequency stimulation of the rabbit tibialis anterior muscle represents an established model system for studying the response of fast fibres to enhanced neuromuscular activity under conditions of maximum activation. We have conducted a DIGE analysis of unstimulated control specimens versus 14- and 60-day conditioned muscles. A differential expression pattern was observed for 41 protein species with 29 increased and 12 decreased muscle proteins. Identified classes of proteins that are changed during the fast-to-slow transition process belong to the contractile machinery, ion homeostasis, excitation-contraction coupling, capillarization, metabolism and stress response. Results from immunoblotting agreed with the conversion of the metabolic, regulatory and contractile molecular apparatus to support muscle fibres with slower twitch characteristics. Besides confirming established muscle elements as reliable transition markers, this proteomics-based study has established the actin-binding protein cofilin-2 and the endothelial marker transgelin as novel biomarkers for evaluating muscle transformation.

Proteomic profiling of pathological and aged skeletal muscle fibres by peptide mass fingerprinting (Review).

In contrast to the traditional biochemical study of single proteins or isolated pathways in health and disease, technical advances in the high-throughput screening of peptides by mass spectrometry have established new ways of identifying entire cellular protein populations in one swift analytical approach. This review discusses the recent progress in the biochemical analysis of skeletal muscle extracts and outlines the mass spectrometry-based proteomics approach for studying muscle tissues in normal and pathobiochemical processes using peptide mass fingerprinting. Individual topics covered include the most commonly inherited muscle disease, X-linked muscular dystrophy, the physiological process of fast-to-slow fibre transformation, and the role of fibre degeneration in age-related muscle wasting. Recent proteomic profiling studies of dystrophic muscles have revealed new disease markers in dystrophin-deficient fibres, such as adenylate kinase, the Ca2+-binding protein regucalcin and the small heat shock protein cvHSP. Since these muscle proteins are of low abundance, they have not previously been identified as biomarkers of muscular dystrophy, illustrating the increased sensitivity of modern mass spectrometric techniques. This review outlines comparative proteomic techniques that employ conventional labeling methods, such as Coomassie- or silver-staining. In addition, the most advanced proteomic screening approach currently available, fluorescence difference in-gel electrophoresis, is described and its potential for studying muscle proteomes is critically examined. As an alternative suggestion, the two-dimensional analysis of different protein samples separated in parallel on a single second dimension gel is introduced and the usefulness of this technique for direct comparative investigations is explained. The potential of studying protein complex formation by intraproteomics, estimating the composition of subcellular fraction by subproteomics, and analyzing total muscle protein extracts by mass spectrometry-based proteomics, is enormous. Proteomics is one of the most promising new analytical ways of comparing large muscle protein complements and has the potential to decisively improve modern biochemical and biomedical research into neuromuscular disorders.

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.

Reduced expression of regucalcin in young and aged mdx diaphragm indicates abnormal cytosolic calcium handling in dystrophin-deficient muscle.

The cytosolic Ca2+ -binding protein regucalcin is involved in intracellular signaling and present in high abundance in the liver. Here, we could show by comparative mass spectrometry-based proteomics screening of normal versus dystrophic fibres that regucalcin of 33.9 kDa and pI5.2 also exists in diaphragm muscle. Since the expression of sarcolemmal Ca2+ -leak channels and luminal Ca2+ -binding elements is altered in dystrophin-deficient muscle, we initiated this study in order to determine whether additional soluble muscle proteins involved in Ca2+ -handling are affected in muscular dystrophy. Following separation by two-dimensional gel electrophoresis, the spot pattern of the normal versus the mdx diaphragm muscle proteome was evaluated by densitometry. The expression levels of 20 major protein spots were shown to change and their identity determined by mass spectrometry. A 2-fold reduction of regucalcin in mdx diaphragm, as well as in dystrophic limb muscle and heart, was confirmed by immunoblotting in both young and aged mdx mice. The results from our proteomics analysis of dystrophic diaphragm support the concept that abnormal Ca2+ -handling is involved in x-linked muscular dystrophy. The reduction in key Ca2+ -handling proteins may result in an insufficient maintenance of Ca2+ -homeostasis and an abnormal regulation of Ca2+ -dependent enzymes resulting in disturbed intracellular signaling mechanisms in dystrophinopathies.

Differential expression of the fast skeletal muscle proteome following chronic low-frequency stimulation.

Physiological and biochemical responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation can be studied experimentally by chronic low-frequency stimulation of fast muscles. Stimulation-induced changes in the expression pattern of the rabbit fast skeletal muscle proteome were evaluated by two-dimensional gel electrophoresis and compared to the altered isoform expression profile of established transformation markers such as the Ca2+-ATPase, calsequestrin and the myosin heavy chain. Sixteen muscle proteins exhibited a marked change in their expression level. This included albumin with a 4-fold increase in abundance. In contrast, glycolytic enzymes, such as enolase and aldolase, showed a decreased expression. Concomitant changes were observed with marker elements of the contractile apparatus. While the fast isoforms of troponin T and myosin light chain 2 were drastically down-regulated, their slow counterparts exhibited increased expression. Interestingly, mitochondrial creatine kinase expression increased while the cytosolic isoform of this key muscle enzyme decreased. The expression of the small heat shock protein HSP-B5/alphaB-crystallin and the oxygen carrier protein myoglobin were both increased 2-fold following stimulation. The observed changes indicate that the conversion into fatigue-resistant red fibres depends on: (i) the optimum utilization of free fatty acids via albumin transportation, (ii) a rearrangement of the creatine kinase isozyme pattern for enhanced mitochondrial activity, (iii) an increased availability of oxygen for aerobic metabolism via myoglobin transport, (iv) the conversion of the contractile apparatus to isoforms with slower twitch characteristics and (v) the up-regulation of chaperone-like proteins for stabilising myofibrillar components during the fast-to-slow transition process.

Early effects of denervation on Ca(2+)-handling proteins in skeletal muscle.

The adaptive response of skeletal muscle fibres depends on a variety of biological factors including loading conditions and neuromuscular activity. An extreme type of atrophy-inducing change in contractile activity is represented by the physical disconnection between the motor nerve and its respective fibre unit. Since fibre type alterations have a striking effect on the Ca(2+)-regulatory apparatus, we have investigated the fate of a key Ca(2+)-pump and essential Ca(2+)-binding proteins in extensor digitorum longus specimens two weeks after nerve crush or complete denervation. In contrast to increased levels of sarcalumenin, immunoblotting revealed that the expression of the fast SERCA1 Ca(2+)-ATPase isoform is drastically decreased and fast calsequestrin is slightly reduced. Analysis of myosin heavy chain isoforms agreed with this result and showed a fast-to-slow fibre type shifting process following denervation. Hence, changes in muscle activity appear to have a profound effect on the abundance and isoform expression pattern of Ca(2+)-handling elements.