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.     

THE RESOLUTION OF CHLOROPHYLL a/b BINDING PROTEINS BY A PREPARATIVE METHOD BASED ON FLAT BED ISOELECTRIC FOCUSING

We describe a new fractionation method for intrinsic membrane proteins based on flat bed isoelectric focusing (IEF) in granulated gel. The characteristics of the separation in the presence of the non-ionic detergent dodecylmaltoside are considered. The method has been applied to the fractionation of chlorophyll a/b binding proteins from chloroplast grana membranes. Several Light Harvesting Complexes II (LHC II) have been resolved showing differences in their polypeptide composition. Probing with monoclonal and polyclonal antibodies showed that polypeptides belonging to different [EF fractions with the same mobility in denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis, are immunologically distinct polypeptides. This is the first report of the presence in the thylakoid membrane of a number of LHCII polypeptides that may reflect the genetic complexity of the Cab genes. Moreover preparative amounts have been obtained of the minor chlorophyll a/b proteins CP 29, CP 26 and CP 24 that have been recently described. The analysis of a currently used LHCII preparation by the present method shows that this fraction is actually contaminated by two minor chlorophyll a/b proteins.     

Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins.

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.     

High-performance size-exclusion chromatography of polypeptides on a TSK G2000SW column in acidic mobile phases

Polypeptides subjected to high-performance liquid chromatography on a TSK G2000SW column in acidic mobile phases of low ionic strength underwent a combination of size exclusion and ion exclusion from the matrix. The distribution coefficient (Kd) of each polypeptide was dependent upon the concentration of ion-pairing agent present and increased as the concentration of ion-pairing agent increased. The fractionation range of the column could thus be manipulated by altering the concentration of acid in the mobile phase. The nature of the ion-pairing acid, in terms of hydrophilic or hydrophobic ion pairing, also had an effect on the chromatography, such that hydrophobic ion-pairing agents lead to increased Kd values, especially in the case of the most basic polypeptides. To account for these results it is proposed that the TSK GSW matrix is cationic at low pH, and that ion pairing suppresses the ion exclusion experienced by cationic polypeptides under these conditions.     

Development of a simple ampholyte-free isoelectric focusing slab electrophoresis for protein fractionation

Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, isoelectric focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. beta-Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H(+) and OH(-) ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The focusing process was visually observed and the separation was confirmed by capillary isoelectric focusing (cIEF) with pI markers.     

BOP: A basic phenylalanine‐rich oligo‐peptide located on the surface of glycolate oxidase influences its pI values

Glycolate oxidase (GO) consists of identical subunits and therefore should show one definite pI value, but the isolated GO exhibited a range of pIs. This study investigated the underlying cause of this phenomenon. GO was purified and showed a molecular weight of 40 kDa by SDS‐PAGE. Elution behavior on DEAE‐cellulose chromatography and cellulose acetate membrane electrophoresis indicated that the purified GO was highly basic (pI>10.0). Repeated IEF and cIEF analysis showed that the pI of the purified GO was in the range of 10.0–3.25, either in a smear form or as distinct bands. 2‐DE analysis showed that the 40 kDa subunit of GO displayed variable pIs from 9.6 to 3.65. It was likely that the purified GO was actually a complex consisted of GO and an unknown protein. CE‐SDS, SDS‐cellulose acetate membrane electrophoresis and amino acid compositions indicated that the unknown protein was a highly basic polymer (BP) consisting of basic and phenylalanine‐rich oligo‐peptide (BOP). Many BOPs are located on the surface of the acidic GO via ionic and hydrophobic interactions and formed GO‐BOP complex (GC), resulting in a highly basic GC although GO itself was acidic. This hypothesis was further supported by the facts that anti‐GC serum failed to recognize GO, and GC showed a peak at 257 nm although GO has few phenylalanine residues. Irregular and incomplete disassociation between GO and BOP was observed in IEF and cIEF, resulting in various intermediates with different ratios of GO/BOP, which could be the reason for the range of pIs observed for GO.     

Mechanisms of insertion of tail-anchored proteins into the membrane of the endoplasmic reticulum

Tail-anchored proteins (TAPs) are a subclass of type II integral membrane proteins that carry out important and diverse functions within cells. Structurally, TAPs present an N-terminal domain exposed to the cytosol and a single transmembrane domain (TMD) close to the C-terminus, the latter is responsible for the targeting and insertion into the proper intracellular membrane (endoplasmic reticulum (ER), mitochondria, peroxisomes). Due to this particular topology, TAPs insert obligatorily into membranes by post-translational pathways and are excluded from the classical SRP dependent co-translational ER insertion. ER-targeted TAPs can follow two distinct ways of insertion according to the hydrophobicity of their TMD. In the "assisted" pathway, TAPs with more hydrophobic TMDs insert in the ER membrane with the requirement of energy and the involvement of proteinaceous component(s). By contrast neither energy, nor membrane or cytosolic proteins are necessary and do not even improve the "unassisted" insertion of TAPs with moderately hydrophobic TMDs. In this review, we discuss the most relevant recent data regarding the molecular mechanism that underlies these processes.     

Correlation of migration behavior in free-flow zone electrophoresis and electrophoretic titration curve

The correlation of electrophoretic migration behavior in free-flow zone electrophoresis (FFZE) and electrophoretic titration curve (ETC) has been explored. It is shown that the ETC of a protein or a mixture of proteins can be used to predict the fraction numbers at which those proteins elute in a preparative scale FFZE experiment. The ETC is a quick and effective way to choose optimal buffer conditions in FFZE. FFZE is employed to determine the isoelectric points (pI) of proteins whose pIs lie beyond the range of IEF 3-9 gels. It is found that separations in FFZE are governed by the net surface charge of the proteins.     

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.     

Separation of proteins using a novel two-depth miniaturized free-flow electrophoresis device with multiple outlet fractionation channels

A novel free-flow electrophoresis glass chip design with two-depth etched structures for the separation and fractionation of proteins is presented. The microfluidic structures etched in two depths enhance the flow characteristics inside the miniaturized device. A novel nine-port outlet interface enables the fractionation of the separated analytes. The separation and focussing of a protein sample mixture demonstrated the ability of the new chip.     

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.     

Proteins of the hepatoma tissue culture cell plasma membrane.

The specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5'-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.     

A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry

When electrospray ionisation mass spectrometry (ESI-MS) is used on-line with capillary isoelectric focusing (CIEF), the presence of the carrier ampholytes creating the IEF pH gradient is not desirable. With the purpose of removing these ampholytes, we have developed a free-flow electrophoresis (FFE) device and coupled it to CIEF. The different parameters inherent to the resulting CIEF/FFE system were optimised using ultraviolet absorbance (UV) detection. The on-line coupling of this system with ESI-MS was successfully realised for three model proteins (myoglobin, carbonic anhydrase I and beta-lactoglobulin B).     

Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e. 1D SDS-PAGE, 1D preparative SDS-PAGE, IEF-IPG, and 2D-PAGE in their performance as fractionation approaches in nano LC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS-PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2D-PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS-PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput.     

High resolution two-dimensional gel analysis of proteins in the central nervous system of larvae of Drosophila melanogaster.

An improved method of high resolution two-dimensional gel electrophoresis was used to study the patterns of protein synthesis in the central nervous system (CNS) of late instar larvae of Drosophila melanogaster. A small number of CNSs was radiolabeled with a mixture of 14C-labeled amino acids or with [35S]methionine, and the pattern of labeled proteins was analyzed. One thousand and forty-five polypeptides, 800 acidic (IEF) and 245 basic (NEPHGE), from the CNS of several wild-type strains have so far been separated and cataloged. All these polypeptides were numbered and compared with our catalog of polypeptides from wing imaginal discs, and quantitative or qualitative changes were detected in more than 23% of the polypeptides. When comparing patterns of label of CNSs we found small quantitative differences in the rate of synthesis between individuals of the same strain, not due to sexual differences, and a minute number of quantitative and qualitative differences between groups of individuals of different strains.     

Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique—isoelectric focusing of proteins and a single-stranded DNA fragment

Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (μFFE) chip with a filling capacity of 9.5 μL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm⁻¹ and 422 V cm⁻¹, depending on the application.     

Miniaturizing free-flow electrophoresis - a critical review

Free-flow electrophoresis (FFE) separation methods have been developed and investigated for around 50 years and have been applied not only to many types of analytes for various biomedical applications, but also for the separation of inorganic and organic substances. Its continuous sample preparation and mild separation conditions make it also interesting for online monitoring and detection applications. Since 1994 several microfluidic, miniaturized FFE devices were developed and experimentally characterized. In contrast to their large-scale counterparts microfluidic FFE (mu-FFE) devices offer new possibilities due to the very rapid separations within several seconds or below and the requirement for sample volumes in the microliter range. Eventually, these mu-FFE systems might find application in so-called lab-on-a-chip devices for real-time monitoring and separation applications. This review gives detailed information on the results so far published on mu-FFE chips, comprising its four main modes, namely free-flow zone electrophoresis (FFZE), free-flow IEF (FFIEF), free-flow ITP (FFITP), and free-flow field-step electrophoresis (FFFSE). The principles of the different FFE modes and the basic underlying theory are given and discussed with special emphasis on miniaturization. Different designs as well as fabrication methods and applied materials are discussed and evaluated. Furthermore, the separation results shown indicate that similar separation quality with respect to conventional FFE systems, as defined by the resolution and peak capacity, can be achieved with mu-FFE separations when applying much lower electrical voltages. Furthermore, innovations still occur and several approaches for hyphenated, more integrated systems have been proposed so far, some of which are discussed here. This review is intended as an introduction and early compendium for research and development within this field.     

Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations

Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains.     

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.     

A review on preparative and semi-preparative offgel electrophoresis for multidimensional protein/peptide assessment

Mass spectrometry (MS) techniques are commonly used for protein identification and further analysis of selected protein spots after high resolution 2-D electrophoresis. Complementary gel-free approaches have been developed during the last few years and have shown to be useful tools in modern proteomics. The development and application of various gel-free electrophoresis devices for performing protein fractionation according to the pI differences is therefore a topic of interest. This review describes the current state of isoelectric focusing (IEF) gel-free electrophoresis based on the Agilent offgel 3100 fractionator. The review includes, therefore, (i) an overview on IEF as well as other previous IEF gel-free electrophoresis developments; (ii) offgel fundamentals and future trends; (iii) advantages and disadvantages of current offgel procedures; (iv) requirements of isolated protein pellets for further offgel fractionation; (v) offgel fraction requirements to perform the second dimensional analysis by advance electrophoresis and chromatographic techniques; and (vi) effect of the offgel operating conditions on the stability of metal–protein complexes.