Proteomic analysis of the atrophying rat soleus muscle following denervation

A proteomic analysis was performed comparing normal rat soleus muscle to denervated soleus muscle at 0.5, 1, 2, 4, 6, 8 and 10 days post denervation. Muscle mass measurements demonstrated that the times of major mass changes occurred between 2 and 4 days post denervation. Proteomic analysis of the denervated soleus muscle during the atrophy process demonstrated statistically significant (at the p < 0.01 level) changes in 73 soleus proteins, including coordinated changes in select groups of proteins. Sequence analysis of ten differentially regulated proteins identified metabolic proteins, chaperone and contractile apparatus proteins. Together these data indicate that coordinated temporally regulated changes in the proteome occur during denervation-induced soleus muscle atrophy, including changes in muscle metabolism and contractile apparatus proteins.     

Ubiquitin Ligases at the Heart of Skeletal Muscle Atrophy Control

Skeletal muscle loss is a detrimental side-effect of numerous chronic diseases that dramatically increases mortality and morbidity. The alteration of protein homeostasis is generally due to increased protein breakdown while, protein synthesis may also be down-regulated. The ubiquitin proteasome system (UPS) is a master regulator of skeletal muscle that impacts muscle contractile properties and metabolism through multiple levers like signaling pathways, contractile apparatus degradation, etc. Among the different actors of the UPS, the E3 ubiquitin ligases specifically target key proteins for either degradation or activity modulation, thus controlling both pro-anabolic or pro-catabolic factors. The atrogenes MuRF1/TRIM63 and MAFbx/Atrogin-1 encode for key E3 ligases that target contractile proteins and key actors of protein synthesis respectively. However, several other E3 ligases are involved upstream in the atrophy program, from signal transduction control to modulation of energy balance. Controlling E3 ligases activity is thus a tempting approach for preserving muscle mass. While indirect modulation of E3 ligases may prove beneficial in some situations of muscle atrophy, some drugs directly inhibiting their activity have started to appear. This review summarizes the main signaling pathways involved in muscle atrophy and the E3 ligases implicated, but also the molecules potentially usable for future therapies.     

Electrophoresis of serum isoenzymes and proteins following acute myocardial infarction

The clinical significance of the serum enzymes creatine kinase (CK, EC 2.7.3.2), lactate dehydrogenase (LD, EC 1.1.1.27) and aspartate aminotransferase (EC 2.6.1.1), and the isoenzymes CK 1-3 and LD 1-5, in acute myocardial infarction (AMI) is reviewed. Particular attention is given to electrophoretic analysis of the isoenzymes (and the CK isoforms/subforms) following AMI and thrombolytic therapy. Other protein markers for the monitoring of AMI, including myoglobin and muscle contractile proteins, are also discussed and the potential for the detection of new marker proteins using high-resolution two-dimensional electrophoretic methods is demonstrated. Whilst emphasis is placed upon electrophoretic methods the value of complementary immunoassays is acknowledged in order to maintain a balanced perspective.     

Erythrocyte troponin inhibitor-like protein: isolation and characterization.

A protein was isolated from a human erythrocyte lysate with an apparent molecular weight of 23,000--24,000 daltons. This protein was purified by batch DEAE cellulose followed by column DEAE cellulose chromatography and a gradient of NaCl. On sodium dodecyl sulfate acrylamide electrophoresis, the erythrocyte protein comigrated with muscle troponin inhibitor. An isoelectric precipitation (pH 9.25) was used for the separation of muscle troponin inhibitor from a complex with another troponin component. Both the erythrocyte protein and the muscle troponin inhibitor partially inhibited muscle myosin Ca2+ and K+-EDTA ATPase activity. Furthermore, they inhibited actin-activated Mg2+-ATPase of muscle myosin. The inhibitory effects were absent in the presence of muscle troponin calcium-binding component. Muscle troponin inhibitor and the erythrocyte troponin inhibitor-like protein bound to muscle myosin when myosin was precipitated twice at low ionic strength. The presence of a troponin inhibitor-like protein in erythrocytes suggests that it may be a component in the regulation of contractile activity.     

An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresis

Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes.     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

High-performance capillary electrophoresis of meat, dairy, and cereal proteins

Food proteins play important roles in food functionality, nutrition, and human health. For these reasons, new analytical methods are continually being developed to separate and characterize these important proteins. High-performance capillary electrophoresis (HPCE) is one of the latest analytical methods to be applied to the separation of food proteins. This review covers methods and applications for the separation of three major groups of food proteins, meat, dairy, and cereal proteins.     

Guanidine affects differentially the twitch response of diaphragm, extensor digitorum longus and soleus nerve-muscle preparations of mice

Guanidine has been used with some success to treat myasthenia gravis and myasthenic syndrome because it increases acetylcholine release at nerve terminals through K?, Na? and Ca2? channels-involving mechanisms. Currently, guanidine derivatives have been proposed for treatment of several diseases. Studies aimed at providing new insights to the drug are relevant. Experimentally, guanidine (10 mM) induces on mouse phrenic nerve-diaphragm (PND) preparations neurotransmission facilitation followed by blockade and a greatest secondary facilitation after its removal from bath. Herein, we hypothesized that this peculiar triphasic response may differ in muscles with distinct twitch/metabolic characteristics. Morphological alterations and contractile response of PND, extensor digitorum longus (EDL) and soleus (SOL) preparations incubated with guanidine (10 mM) for 15, 30, 60 min were analyzed. Guanidine concentrations of 5 mM (for PND and EDL) and 1 mM (for EDL) were also tested. Guanidine triphasic effect was only observed on PND regardless the concentration. The morphological alterations in muscle tissue varied along time but did not impede the PND post-wash facilitation. Higher doses (20-25 mM) did not increase EDL or SOL neurotransmission. The data suggest a complex mechanism likely dependent on the metabolic/contractile muscle phenotype; muscle fiber types and density/type of ion channels, sarcoplasmic reticulum and mitochondria organization may have profound impact on the levels and isoform expression pattern of Ca2? regulatory membrane proteins so reflecting regulation of calcium handling and contractile response in different types of muscle.     

Cerium-mediated Free Radicals in Contractile Proteins. EPR and DSC study

The effect of free radicals obtained in hydroxyl and cerium(IV)-nitrilotriacetic acid free radical generating systems on contractile proteins (actin, myosin and their complexes in glycerinated muscle fibres) was studied using differential scanning calorimetry and spin trapping electron paramagnetic resonance technique. The analysis of spectra showed that selective attack of thiol groups – Cys-257 and Cys-374 residues of actin, and among others Cys-707 residue of myosin – and random attack of sidechains of the main proteins of muscle tissue produced structural and functional changes, which affected the ATP hydrolysis cycle and very likely the dynamics of actin. The melting curves obtained on protein systems support the view that global conformational changes accompany the local damage of free radicals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Trennungen von einigen Proteinen und deren Peptiden aus Rindermuskel mit Hilfe der Dünnschicht-Fingerprint-Technik

A report is given on the application of the thin-layer fingerprint technique with cellulose powder MN 300 to the separation of peptides isolated from beef muscle proteins and on free peptides and amino-acids of egg powders. The method allows a separation of substance with relative small and sharp limited spots.     

Separation and identification of photosynthetic antenna membrane proteins by high- performance liquid chromatography electrospray ionization mass spectrometry

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.     

Two-dimensional protein map of human vastus lateralis muscle

Protein samples of human vastus lateralis muscle were analyzed by two-dimensional gel electrophoresis, using immobilized pH gradients encompassing several pH regions in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. More than 500 protein spots on each gel were detected by silver staining, of which 150 were excised, digested in-gel with trypsin and characterized by matrix assisted laser desorptioin/ionization-mass spectrometry and tandem electrospray mass spectrometry. Using these techniques, 124 spots including contractile proteins and metabolic enzymes were identified. This database should provide a valuable resource for the definition of the functional properties of muscle fibres and for assessment of the influence of e.g., hypoxia, ischaemia and ageing on protein level.     

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.     

Protein- versus peptide fractionation in the first dimension of two-dimensional high-performance liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry for qualitative proteome analysis of tissue samples

The availability of robust and highly efficient separation methods represents a major requirement for proteome analysis. This study investigated the characteristics of two different gel-free proteomic approaches to the fractionation of proteolytic peptides and intact proteins, respectively, in a first separation dimension. Separation and mass spectrometric detection by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) were performed at the peptide level in both methods. Bottom-up analysis (BU) was carried out employing well established peptide fractionation in the first separation dimension by strong cation-exchange chromatography (SCX), followed by ion-pair reversed-phase chromatography (IP-RPC) in the second dimension. In the semi-top-down approach (STD), which involved intact protein fractionation in the first dimension, the separation mode in both dimensions was IP-RPC utilizing monolithic columns. Application of the two approaches to the proteome analysis of proteins extracted from a tumor tissue revealed that the BU method identified more proteins (1245 in BU versus 920 in STD) while STD analysis offered higher sequence coverage (14.8% in BU versus 17.5% in STD on average). The identification of more basic and larger proteins was slightly favored in the BU approach, most probably due to higher losses of these proteins during intact protein handling and separation in the STD method. A significant degree of complementarity was revealed by an approximately 33% overlap between one BU and STD replicate, while 33% each of the protein identifications were unique to both methods. In the STD method, peptides obtained upon digestion of the proteins contained in fractions of the first separation dimension covered a broad elution window in the second-dimension separation, which demonstrates a high degree of “pseudo-orthogonality” of protein and peptide separation by IP-RPC in both separation dimensions.     

A comparison of Tris-glycine and Tris-tricine buffers for the electrophoretic separation of major serum proteins

This paper compares different buffer systems for the electrophoretic separation of the five most abundant serum proteins on native-PAGE gel and cellulose membranes. A modified Tris-tricine system was shown to be superior for the separation of these serum proteins in a 7% m/v native-PAGE gel as compared with the traditionally used Tris-glycine and Tris-tricine methods. This modified Tris-tricine buffer system was also employed for the separation of serum proteins using a cellulose acetate membrane and very effective separation was observed as compared with the traditionally used Tris-barbital and Tris-glycine buffer systems.     

Electrophoretic separation method for membrane pore‐forming proteins in multilayer lipid membranes

In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore‐forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high‐performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore‐forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore‐forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore‐forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high‐performance by utilizing the specific properties of lipid membranes.     

Shotgun proteomic analysis of Arabidopsis thaliana leaves

Two shotgun tandem MS proteomics approaches, multidimensional protein identification technology (MudPIT) and 1-D gel-LC-MS/MS, were used to identify Arabidopsis thaliana leaf proteins. These methods utilize different protein/peptide separation strategies. Detergents not compatible with MudPIT were used with 1-D gel-LC-MS/MS to help enrich for the detection of membrane-spanning and hydrophobic proteins. By combining the data from all MudPIT and 1-D gel-LC-MS/MS experiments, 2342 nonredundant proteins spanning a broad range of molecular weights and pI values were detected. With the exception of unknown proteins, the distribution of gene ontology (GO) classifications for the detected proteins was similar to that encoded by the genome, which shows that these extraction and separation procedures are useful for a broad proteomic survey of plant cells. Unknown proteins will likely have to be targeted by using additional methods, some of which should be compatible with separation strategies taken here.     

一种规模化蛋白质组分离和鉴定新方法研究及其应用

建立了一种规模化的蛋白质组分离和鉴定新方法。通过对在生命发育过程中具有重要研究价值的人胎肝线粒体蛋白质组的分离分析，表明与毛细管液-质联用的不同分离方法的组合可以增大检测动态范围和分辨率。研究共鉴定了2977个肽段，归属于915种蛋白质。去除批次间冗余后，鉴定的蛋白质为477种，其中291种为唯一蛋白质，186种为蛋白质簇，144种蛋白质明确定位于人胎肝线粒体中。所鉴定蛋白质的分子量分布范围为7000Da～330000 Da，pI值分布在4．0～11．89，克服了两维凝胶电泳在分子量和pH方面的歧视性问题。实验中发现的蛋白质簇以及确定一种蛋白质需要最少肽段数的问题还需要进一步研究。     

Comparison of the separation of proteins of wide-ranging molecular weight via trilobal polypropylene capillary-channeled polymer fiber,commercial superficiously porous,and commercial size exclusion columns

Reversed phase and size-exclusion chromatography methods are commonly used for protein separations, although they are based on distinctly different principles. Reversed phase methods yield hydrophobicity-based (loosely-termed) separation of proteins on porous supports, but tend to be limited to proteins with modest molecular weights based on mass transfer limitations. Alternatively, size-exclusion provides complementary benefits in the separation of higher mass proteins based on entropic, not enthalpic, processes, but tend to yield limited peak capacities. In this study, microbore columns packed with a novel trilobal polypropylene capillary-channeled polymer fiber were used in a reversed phase modality for the separation of polypeptides and proteins of molecular weights ranging from 1.4 to 660 kDa. Chromatographic parameters including gradient times, flow rates, and trifluoroacetic acid concentrations in the mobile phase were optimized to maximize resolution and throughput. Following optimization, the performance of the trilobal fiber column was compared to two commercial-sourced columns, a superficially porous C4-derivatized silica and size exclusion, both of which are sold specifically for protein separations and operated according to the manufacturer-specified conditions. In comparison to the commercial columns, the fiber-based column yielded better separation performance across the entirety of the suite, at much lower cost and shorter separation times.     

High-performance liquid chromatography and capillary electrophoresis for the analysis of maize proteins

Methods for the analysis of maize proteins using HPLC and CE are reviewed. Most of the references cited in this review concern HPLC methods. Size-exclusion HPLC and especially RP-HPLC methods have been developed for characterization of normal and genetically modified maize, cultivar differentiation, and prediction of quality. Few CE methods for the analysis of maize proteins were found in the existing literature. Most of these methods focus on optimization of the separation of maize proteins using CZE and SDS-capillary gel electrophoresis.