High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.     

LC‐MS/MS as an alternative for SDS‐PAGE in blue native analysis of protein complexes

Two‐dimensional blue native/SDS‐PAGE is widely applied to investigate native protein–protein interactions, particularly those within membrane multi‐protein complexes. MS has enabled the application of this approach at the proteome scale, typically by analysis of picked protein spots. Here, we investigated the potential of using LC‐MS/MS as an alternative for SDS‐PAGE in blue native (BN) analysis of protein complexes. By subjecting equal slices from BN gel lanes to label‐free semi‐quantitative LC‐MS/MS, we determined an abundance profile for each protein across the BN gel, and used these profiles to identify potentially interacting proteins by protein correlation profiling. We demonstrate the feasibility of this approach by considering the oxidative phosphorylation complexes I–V in the native human embryonic kidney 293 mitochondrial fraction, showing that the method is capable of detecting both the fully assembled complexes as well as assembly/turnover intermediates of complex I (NADH:ubiquinone oxidoreductase). Using protein correlation profiling with a profile for subunits NDUFS2, 3, 7 and 8 we identified multiple proteins possibly involved in the biogenesis of complex I, including the recently implicated chaperone C6ORF66 and a novel candidate, C3ORF60.     

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes

The recent upsurge in proteomics research has been facilitated largely by streamlining of two-dimensional (2-D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2-D gels. In addition, functional information regarding protein:protein interactions is lost during 2-D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2-D blue-native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed.     

Phosphoprotein profiling by PA-GeLC-MS/MS

A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.     

A DIGE-based approach to study interacting proteins

A full spectrum of high-throughput protein identification and characterization approaches has been developed for protein profiling. However, the most demanding field to better understanding protein interactions known as the "interactome" is still of a perpetual need for modern proteomics. Recently developed DIGE (difference in-gel electrophoresis) system may be of potential use when studying interacting proteins. In this work we applied DIGE technique on native gel electrophoresis to study protein-protein interactions. As a proof of principle, we utilized an in vitro interaction model between p53 and HDM2 proteins. In parallel, we also showed interaction of these proteins using fluorescently labelled p53- or HDM2-immunoprecipitation pellets. Thus, we believe this study shows a good potential for investigating various interacting partners and benefits towards creation of interactome.     

Extraction of proteins from the large subunit of bovine mitochondrial ribosomes under nondenaturing conditions

The 55 S mammalian mitochondrial ribosome (referred to hereafter as "mitoribosome") is protein-rich, containing nearly twice as much protein as the Escherichia coli ribosome. In order to produce soluble mitochondrial proteins and protein-deficient subribosomal particles for use in functional and structural studies, the proteins of bovine mitoribosomes were extracted by washing in a series of buffers containing increasing concentrations of LiCl as the only chaotropic agent. LiCl disruption is used in order to preserve the solubilized proteins in a substantially "native" configuration. The extraction mixtures were characterized by sucrose density gradient analysis and the compositions of the stripped protein and residual pellet fractions were determined by two-dimensional polyacrylamide gel electrophoresis. In order to analyze the behavior or individual proteins, the intensity of Coomassie blue stain for each protein was normalized against the intensity of stain for the same protein in a control sample. Buffers with 1, 2, and 4 M LiCl each extract a specific subset of mitoribosomal proteins, while another group of proteins remains in the residual pellet fraction. Although very few proteins are detected in only one condition, most proteins are specifically enriched in one fraction. This LiCl procedure, therefore, produces fractionated groups of mitoribosomal proteins which can be used directly as a source for those proteins in which they are enriched, or they can be used as a starting point in further purification procedures. In contrast to results with E. coli ribosomes, several mitoribosomal proteins remain core-associated, indicating a different structural organization in these ribosomes.     

Optimization of the scratch assay for in vitro skeletal muscle wound healing analysis

To isolate mitochondrial complexes, we have combined elements from the classic Laemmli protocol and blue native polyacrylamide gel electrophoresis (BN–PAGE) methods to develop a straightforward modified native electrophoresis protocol. This modified protocol presented good resolution for native electrophoresis of inner mitochondrial membrane proteins, where bands were easily visualized with no leftover stain or gel lanes overlap. Enzymatic tests revealed that complexes I and V remain active in the gel. This protocol, designed to overcome specific limitations of the standard protocols, provides a potential methodology to study membrane proteins in their functional form.     

Proteomics: quantitative and physical mapping of cellular proteins

Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function. Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process. The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level. Proteomics can be divided into expression proteomics, the study of global changes in protein expression, and cell-map proteomics, the systematic study of protein-protein interactions through the isolation of protein complexes.     

Features and applications of blue-native and clear-native electrophoresis

1-D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein-protein interactions can be identified depending on detergent and buffer conditions. 1-D native gels are immediately applicable for in-gel detection of fluorescent-labeled proteins and for in-gel catalytic activity assays. 1-D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1-D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2-D separation of subunits of protein complexes by SDS-PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, and the vast variety of mitochondrial encephalomyopathies.     

Identification and Analysis of Multi-Protein Complexes in Placenta

Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders.     

Chronic ethanol feeding causes depression of mitochondrial elongation factor Tu in the rat liver: implications for the mitochondrial ribosome

Chronic ethanol feeding is known to negatively impact hepatic energy metabolism. Previous studies have indicated that the underlying lesion responsible for this may lie at the level of the mitoribosome. The aim of this study was to characterize the structure of the hepatic mitoribosome in alcoholic male rats and their isocalorically paired controls. Our experiments revealed that chronic ethanol feeding resulted in a significant depletion of both structural (death-associated protein 3) and functional [elongation factor thermo unstable (EF-Tu)] mitoribosomal proteins. In addition, significant increases were found in nucleotide elongation factor thermo stable (EF-Ts) and structural mitochondrial ribosomal protein L12 (MRPL12). The increase in MRPL12 was found to correlate with an increase in the levels of the 39S large mitoribosomal subunit. These changes were accompanied by decreased levels of nuclear- and mitochondrially encoded respiratory subunits, decreased amounts of intact respiratory complexes, decreased hepatic ATP levels, and depressed mitochondrial translation. Mathematical modeling of ethanol-mediated changes in EF-Tu and EF-Ts using prederived kinetic data predicted that the ethanol-mediated decrease in EF-Tu levels could completely account for the impaired mitochondrial protein synthesis. In conclusion, chronic ethanol feeding results in a depletion of mitochondrial EF-Tu levels within the liver that is mathematically predicted to be responsible for the impaired mitochondrial protein synthesis seen in alcoholic animals.     

The integrated approach to the investigation of soluble multisubunit protein complexes in cyanobacterial cells

Functional proteomics is currently one of the most important tools used for the studying of complex intracellular processes involving a variety of proteins that interact with each other with different probability. Many of them, fulfilling their functions within the complex network of interactions, affect functions of other proteins. Despite the fact that many of the components of these “protein networks” could be previously described individually, it did not give complete details about nature and components of the proposed interactions under natural conditions. The article describes the methods (native gel electrophoresis, size exclusion chromatography, and immuneprecipitation), the most effective in the study of protein interactions and optimized for the study of soluble multisubunit protein complexes in the cells of photosynthetic bacteria using a unicellular cyanobacterium Synechococcus elongates PCC 7942.     

An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes

An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex.     

An alternative strategy to determine the mitochondrial proteome using sucrose gradient fractionation and 1D PAGE on highly purified human heart mitochondria

An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using metrizamide gradients to remove cytosolic and other subcellular contaminant proteins; (b) separation of mitochondrial protein complexes using sucrose density gradients after solubilization with n-dodecyl-beta-D-maltoside; (c) 1D electrophoresis of the sucrose gradient fractions; (d) high-throughput proteomics using robotic gel band excision, in-gel digestion, MALDI target spotting and automated spectral acquisition; and (e) protein identification from mixtures of tryptic peptides by high-precision peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane proteins such as the adenine nucleotide translocase that are not recovered in 2D gels. The technique described here should also be useful for the identification of new protein-protein associations as exemplified by the validation of a recently discovered complex that involves proteins belonging to the prohibitin family.     

Identification of the Toxoplasma gondii mitochondrial ribosome,and characterisation of a protein essential for mitochondrial translation

Apicomplexan parasites cause diseases such as malaria and toxoplasmosis. The apicomplexan mitochondrion shows striking differences from common model organisms, including fundamental processes such as mitochondrial translation. Despite evidence that mitochondrial translation is essential for parasite survival, it is largely understudied. Progress has been restricted by the absence of functional assays to detect apicomplexan mitochondrial translation, a lack of knowledge of proteins involved in the process and the inability to identify and detect mitoribosomes. We report the localization of 12 new mitochondrial proteins, including 6 putative mitoribosomal proteins. We demonstrate the integration of three mitoribosomal proteins in macromolecular complexes, and provide evidence suggesting these are apicomplexan mitoribosomal subunits, detected here for the first time. Finally, a new analytical pipeline detected defects in mitochondrial translation upon depletion of the small subunit protein 35 (TgmS35), while other mitochondrial functions remain unaffected. Our work lays a foundation for the study of apicomplexan mitochondrial translation.     

Unraveling tobacco BY-2 protein complexes with BN PAGE/LC-MS/MS and clustering methods

To understand physiological processes, insight into protein complexes is very important. Through a combination of blue native gel electrophoresis and LC-MS/MS, we were able to isolate protein complexes and identify their potential subunits from Nicotiana tabacum cv. Bright Yellow-2. For this purpose, a bioanalytical approach was used that works without a priori knowledge of the interacting proteins. Different clustering methods (e.g., k-means and hierarchical clustering) and a biclustering approach were evaluated according to their ability to group proteins by their migration profile and to correlate the proteins to a specific complex. The biclustering approach was identified as a very powerful tool for the exploration of protein complexes of whole cell lysates since it allows for the promiscuous nature of proteins. Furthermore, it searches for associations between proteins that co-occur frequently throughout the BN gel, which increases the confidence of the putative associations between co-migrating proteins. The statistical significance and biological relevance of the profile clusters were verified using functional gene ontology annotation. The proof of concept for identifying protein complexes by our BN PAGE/LC-MS/MS approach is provided through the analysis of known protein complexes. Both well characterized long-lived protein complexes as well as potential temporary sequential multi-enzyme complexes were characterized.     

Defining the protein complex proteome of plant mitochondria

A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial "protein complex proteome" of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.     

The photosynthesis apparatus of European mistletoe (Viscum album)

European mistletoe (Viscum album) is known for its special mode of cellular respiration. It lacks the mitochondrial NADH dehydrogenase complex (Complex I of the respiratory chain) and has restricted capacities to generate mitochondrial adenosine triphosphate (ATP). Here, we present an investigation of the V. album energy metabolism taking place in chloroplasts. Thylakoids were purified from young V. album leaves, and membrane-bound protein complexes were characterized by Blue native polyacrylamide gel electrophoresis as well as by the complexome profiling approach. Proteins were systematically identified by label-free quantitative shotgun proteomics. We identified >1,800 distinct proteins (accessible at https://complexomemap.de/va_leaves), including nearly 100 proteins forming part of the protein complexes involved in the light-dependent part of photosynthesis. The photosynthesis apparatus of V. album has distinct features: (1) comparatively low amounts of Photosystem I; (2) absence of the NDH complex (the chloroplast pendant of mitochondrial Complex I involved in cyclic electron transport (CET) around Photosystem I); (3) reduced levels of the proton gradient regulation 5 (PGR5) and proton gradient regulation 5-like 1 (PGRL1) proteins, which offer an alternative route for CET around Photosystem I; (4) comparable amounts of Photosystem II and the chloroplast ATP synthase complex to other seed plants. Our data suggest a restricted capacity for chloroplast ATP biosynthesis by the photophosphorylation process. This is in addition to the limited ATP supply by the mitochondria. We propose a view on mistletoe’s mode of life, according to which its metabolism relies to a greater extent on energy-rich compounds provided by the host trees.

European mistletoe not only has a very special type of cellular respiration, but also an unusual apparatus for the light-dependent part of photosynthesis.     

Electrophoresis techniques to investigate defects in oxidative phosphorylation

Defects in mitochondrial oxidative phosphorylation (OXPHOS) are a frequent cause of severe inherited metabolic disorders and also contribute to aging. The OXPHOS system constitutes five multi-subunit complexes embedded in the mitochondrial inner membrane. Correct function of this system requires proper assembly of the 80 proteins in the complexes, as well as numerous assembly factors. Blue native electrophoresis has become a crucial tool to investigate OXPHOS-related defects in mitochondrial disease patients. In addition, OXPHOS-assembly profiles can be obtained by two dimensional blue native/SDS gel electrophoresis, which provides additional information for identifying disease-causing mutations and insight in the role of specific proteins in the biogenesis of the OXPHOS system. Here we provide a practical guide on how to set-up the basic technique to study OXPHOS defects in patient-derived cells and tissues.     

Solubilization of membrane protein complexes for blue native PAGE

Blue native PAGE is an electrophoretic technique for high-resolution separation of membrane proteins. The method has been proven especially useful for investigation of native protein complexes enabling a characterization of potential protein-protein interactions in the context of functional proteomics. Blue native PAGE is easy to realise, results are reproducible and a high number of protocols are available. However, care should be taken during solubilization of protein complexes to achieve significant results in BN-PAGE analysis. Solubilization of membranes and proteins is not only influenced by detergent-lipid and detergent-protein interactions but also by lipid-lipid, lipid-protein and protein-protein interactions. Interactions have been investigated experimentally and theoretically. But, in practice, the experimental results do not always mirror the theoretical basis and therefore optimal solubilization conditions for each membrane and membrane protein complex should be investigated individually to tap the full potential of BN-PAGE analysis.