LC-nanospray-MS/MS analysis of hydrophobic proteins from membrane protein complexes isolated by blue-native electrophoresis

The application of two-dimensional electrophoresis for the identification of hydrophobic membrane proteins is principally hampered by precipitation of many of these proteins during first-dimension, isoelectric focusing. Therefore new strategies towards the identification and characterization of membrane proteins are being developed. In this work we present a direct and rapid approach from blue-native gels to mass spectrometry, which allows the analyses of complete complexes and prevents protein aggregation of hydrophobic regions during electrophoresis. We combine blue-native gel electrophoresis and liquid chromatography--nanospray-iontrap tandem mass spectrometry to analyze the composition of oxidative phosphorylation complexes I, III, IV and V from bovine-heart mitochondria as a model system containing a number of highly hydrophobic proteins. Bands from blue-native gels were subjected either to in-gel or to in-solution tryptic digestion. The obtained peptide mixtures were further analyzed by liquid chromatography--tandem mass spectrometry and the corresponding proteins were identified by database search. From a total of 86 proteins, 67 protein subunits could be identified including all highly hydrophobic components, except the ND4L and ND6 subunits of complex I. We demonstrate that liquid chromatography--tandem mass spectrometry combined to blue-native electrophoresis is a straightforward tool for proteomic analysis of multiprotein complexes, and especially for the identification of very hydrophobic membrane protein constituents that are not accessible by common isoelectric focusing/sodium dodecyl sulphate gel electrophoresis.     

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.     

Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

Agarose isoelectric focusing can improve resolution of membrane proteins in the two-dimensional electrophoresis of bacterial proteins

2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE.     

Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.     

Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.     

A multichannel gel electrophoresis and continuous fraction collection apparatus for high‐throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ～10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.     

Discontinuous native protein gel electrophoresis

Analysis of the oligomeric state of a native protein usually requires analytical ultracentrifugation or repeated gel filtration to calculate the protein's size. We have developed a discontinuous native protein gel electrophoresis system that allows the separation of even basic proteins according to their size, oligomeric state, and shape. This gel system combines the addition of negative charges to the proteins by Serva Blue G with a discontinuous buffer system and gradient gels. As in SDS-PAGE, chloride constitutes the high mobility anion in the gel and anode buffer. However, for sample focusing this system employs histidine instead of glycine as the slow dipolar ion following from the cathode buffer to improve migration of basic proteins. In addition, proteins run into gel pores corresponding to their size and shape in the gradient gel. Using this gel system, we show that the polypyrimidine tract-binding protein (PTB) is a monomer.     

"LaneSpector", a tool for membrane proteome profiling based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis/liquid chromatography-tandem mass spectrometry analysis: application to Listeria monocytogenes membrane proteins

Proteomics is required to provide insight into any type of subproteome. While the workflow based on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) can be applied for many subproteomes and comprises well-established strategies for data presentation and data analysis, the comprehensive investigation of membrane proteomes remains a challenging task. We present a number of procedures that provide an insight into such systems. We have established a novel protocol for the efficient preparation of membrane fractions, which is used here for the human pathogen Listeria monocytogenes that overcomes difficulties associated with ribosomes. Subsequently, we have used the combination of sodium dodecyl sulfate (SDS)-PAGE and liquid chromatography-tandem mass spectrometry for the characterization of the membrane proteome. Three hundred and one different membrane proteins could be identified, including 70 proteins that exhibited 2-15 transmembrane domains. However, a remarkably high ratio of proteins was detected in gel sections that were not in accordance with their expected migration behavior during SDS-PAGE. Protein identifications based on MASCOT significance criteria could be shown to be of high quality and therefore could not be the explanation of this observation. Consequently we have developed LaneSpector, a general visualization tool that allows the systematic comparison between apparent and calculated protein masses, which is routinely applicable to any high-throughput approach using a mass-dependent separation dimension prior to LC-MS/MS. The detailed presentation of the LaneSpector plot promotes the validation of the analytical process and might help to reveal relevant biological processes such as proteolysis or other post-translational modifications.     

Proteome analysis of Corynebacterium glutamicum.

By the use of different Corynebacterium glutamicum strains more than 1.4 million tons of amino acids, mainly L-glutamate and L-lysine, are produced per year. A project was started recently to elucidate the complete DNA sequence of this bacterium. In this communication we describe an approach to analyze the C. glutamicum proteome, based on this genetic information, by a combination of two-dimensional (2-D) gel electrophoresis and protein identification via microsequencing or mass spectrometry. We used these techniques to resolve proteins of C. glutamicum with the aim to establish 2-D protein maps as a tool for basic microbiology and for strain improvement. In order to analyze the C. glutamicum proteome, methods were established to fractionate the C. glutamicum proteins according to functional entities, i.e., cytoplasm, membranes, and cell wall. Protein spots of the cytoplasmic and membrane fraction were identified by N-terminal sequencing, immunodetection, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). Additionally, a protocol to analyze proteins secreted by C. glutamicum was established. Approximately 40 protein spots were observed on silver-stained 2-D gels, 12 of which were identified.     

Electrophoretic isolation of membrane proteins from acrylamide gels

Protocols for the optimal resolution of membrane and watersoluble proteins in SDS-denatured state (Tricine SDS-PAGE and Blue Tricine SDS-PAGE; Laemmli SDS-PAGE and Blue Laemmli SDSPAGE) and in the native state (Blue Native PAGE) are presented. The protocols for protein recovery from these gels include techniques of electroelution and electroblotting optimized to the type of the preceding electrophoresis system. Native and denatured proteins thus are obtainable in near quantitative yield in soluble and in immobilized form. These techniques can optionally be performed in the milligram range, e.g., for the use of immunization and N-terminal protein sequencing, or in the analytical range.     

Determination of the number and relative molecular mass of subunits in an oligomeric protein by two-dimensional electrophoresis Application to the subunit structure analysis of rat liver amidophosphoribosyltransferase

To determine simultaneously the relative molecular mass (M_r) of a native oligomeric protein, and the number and M_r of its subunits, a method using two-dimensional electrophoresis was developed. To determine the M_r of a native oligomeric protein, pore gradient gel electrophoresis was performed for the first dimension. Native proteins were dissociated into their subunits by sodium dodecyl sulphate (SDS) in a gel slice, then applied to SDS polyacrylamide gel electrophoresis for the second dimension to determine the M_r of subunits. The advantage, accuracy, limitations and application of the method are discussed.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Native multimer analysis of plasma and platelet von Willebrand factor compared to denaturing separation: implication for the interpretation of satellite bands

Blue native electrophoresis (BNE) was applied to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology to perform blue native electrophoresis on human plasma proteins, which has not been done before. The major difference between this method and the commonly used SDS-agarose gel electrophoresis is the lack of satellite bands in the high-resolution native gel. To further analyze this phenomenon, a second dimension was performed under denaturing conditions. Thereby, we obtained a pattern in which each protein sub-unit from the first dimension dissociates into three distinct sub-bands. These bands confirm the triplet structure, which consists of an intermediate band and two satellite bands. By introducing the second dimension, our novel method separates the triplet structure into a higher resolution than the commonly used SDS-agarose gel electrophoresis does. This helps considerably in the classification of ambiguous von Willebrand's disease subtypes. In addition, our method has the additional advantage of being able to resolve the triplet structure of platelet vWF multimers, which has not been identified previously through conventional SDS-agarose electrophoresis multimer analysis. This potential enables us to compare the triplet structure from platelet and plasmatic vWF, and may help to find out whether structural abnormalities concern the vWF molecule in the platelet itself, or whether they are due to the physiological processing of vWF shed into circulation. Owing to its resolution and sensitivity, this native separation technique offers a promising tool for the analysis and detection of von Willebrand disorder, and for the classification of von Willebrand's disease subtypes.     

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.     

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.     

Native polyacrylamide electrophoresis in the presence of Ponceau Red to study oligomeric states of protein complexes

Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.     

Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis

We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures. First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes. The protein complexes are electroeluted and completely detached from Coomasssie blue. Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex). The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes. Furthermore, extremely hydrophobic proteins are detectable on the gels.     

Separation of membrane proteins by two-dimensional electrophoresis using cationic rehydrated strips

Due to their poor solubility during IEF membrane proteins cannot be separated and analyzed satisfactorily with classical 2-DE. A more efficient method for such hydrophobic proteins is the benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE, but the corresponding protocol is intricate and time-consuming. We now developed an easy-to-handle electrophoresis method in connection with a novel device which enables reproducible separation of ionic solubilized membrane proteins using individually rehydrated plastic sheet gel strips. These strips are suitable for the first dimension in a 2-D 16-BAC/SDS system and can be handled easily; this is demonstrated by the separation of membrane proteins of human embryonic kidney (HEK293) cells.     

Capturing sodium dodecyl sulfate-treated protein antigens by antibody affinity electrophoresis

Modifications to antibody affinity electrophoresis for improved detection of proteins have been developed. The bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution for better incorporation of the bait antibody into a distinct region of a 10% w/v polyacrylamide gel. The addition of glutaraldehyde alleviates the need of an electrophoresis buffer with a specific pH. The protein sample to be analyzed is treated with 2% w/v sodium dodecyl sulfate (SDS) to ensure that they carry a negative charge. The negative charge will allow the proteins to migrate towards the cathode and hence pass through the area embedded with the bait antibody. It is observed that electrophoretic migration of bovine serum albumin (BSA) or protein G ceases upon encounter with anti-BSA whereas proteins ovalbumin, beta-lactoglobulin A, and myoglobin migrate freely. However, the addition of 0.1% w/v SDS in the native gel running buffer disrupts the antibody-antigen bond and neither BSA nor protein G can be captured by anti-BSA.