In this other major type of protein separation method, proteins are made to migrate in an electric field, using the fact that they are charged analytes. The various elec-trophoretic setups make use of some structural parameters of the proteins to induce a separation of these proteins. Unlike chromatography, there is theoretically no absolute need for a support, and electrophoresis can be carried out in a liquid vein. However, many electrophoresis techniques perform much better when a gelified support is available. This gelified support combines mechanical strength and easy handling of solids with the liquid phase needed for protein separation. Among these, polyacrylamide gels are the most frequently used, as they combine a good separation versatility with a very low adsorption of proteins.
Because of the movement of ions in the electric field used for driving the separation process, most electrophoretic techniques generate an appreciable amount of Joule heat. This Joule heat induces important thermal and convection problems, which considerably limit the possible scale-up of electrophoresis techniques. Thus, most electrophoretic techniques have been optimized as analytical more than preparative techniques. However, with the recent progress of protein microanalysis techniques and the concomitant decrease in the amount of proteins needed for analysis, the capacity of electrophoretic techniques is now more and more able to deliver the required microquantities of proteins, and so-called "micropreparative" electrophoretic techniques are now more frequently used in proteomics. One of the best examples of this evolution is two-dimensional electrophoresis of proteins, as described in an earlier chapter.
Among the many parameters usable for separating proteins, only three are used in electrophoresis: the protein's charge, size, and isoelectric point. The other parameters (e.g., hydrophobicity) require a reasonable scale of adsorption phenomenon to occur between the protein and some solid support. When adsorption takes place in an electrophoretic process, this induces a strong tailing of the separated zones and thus decreases resolution dramatically. This phenomenon is the plague of capillary electrophoresis, where adsorption of proteins on the walls of the capillary prevents this technique from being widely used as a separation tool at the pro-teomics scale.
Electric Charge as a Separation Parameter in Electrophoresis The basic equations for the electrophoretic movement of analytes in an electric field show that the mobility is proportional to the electric charge of the analyte and to the electric field, and limited by its friction in the separation medium. Thus, the electric charge of proteins could be an interesting separation parameter. However, this is mainly true in liquid media. In gel-based media, the additional friction induced by the gel material makes the friction parameter dominant over the native charge. This means that charge differences result in a smaller separation than size differences. Thus, electric charge is used almost exclusively in capillary electrophoresis and in free-flow electrophoresis. These methods, however, are not very popular at the proteomics scale because of their rather difficult implementation and rather low resolution for proteins. In addition to this role as a driving parameter in elec-trophoresis, the electric charge of the proteins also plays a role in preventing spurious aggregation during separation. Proteins of the same type of charge repel each other, and this electrostatic repulsion is very important, although often unnoticed, in the performances of high-resolution electrophoretic techniques, as will be seen later. Conversely, proteins with opposite charges tend to aggregate and this considerably limits the scope of charge-based electrophoresis techniques.
Size as the Separation Parameter in Electrophoresis: Zone Electrophoresis in Gels As mentioned earlier, size is the dominant separation parameter in elec-trophoretic separations of proteins in gel-based media. It is thus very easy to implement as a separation parameter in electrophoresis techniques. However, the simplest setup (i.e., sample solubilized in the same buffer as the one used for the gels and for the electrode reservoirs and so-called continuous electrophoresis) is a rather low resolution technique due to the fact that diffusion and, to a lesser extent, convection are at play during the migration of the proteins in the electric field. Thus, the thinness of the protein zones at the end of the separation process can only be wider than the one at the beginning of the process. Because it is often difficult to reach important concentrations in protein solutions, the amounts needed for most purposes result in important volumes compared to the scale of the process and thus to wide zones.
The solution to this vexatious problem is the so-called discontinuous electropho-resis setup, which was described by Ornstein and Davis in the mid 1960's (Davis, 1964) and theoretized by Jovin in the early 1970s (Jovin, 1973). This process uses the isotachophoresis process, which states that under proper conditions, ions rank themselves under an electric field by their decreasing mobility with so-called moving boundaries between the various ionic species. When combined with the use of ions whose mobility is strongly pH dependent and can thus be easily modulated by the pH of the gel, and with the flexibility afforded by gelified media, it is possible to devise a setup in which two different steps take place successively. In the first step, called the stacking step, the isotachophoretic conditions between the fast (leading) and slow (trailing) ions are set with a very low mobility. Proteins therefore concentrate at the moving boundary between the leading and trailing ions. This ensures a very strong concentration effect, and thus a very high resolution afterward. In the second step, the speed of the moving boundary is increased, generally by a change in the pH, to a point where proteins are no longer mobile enough to remain in the moving boundary and are therefore separated. While the resolution of the system is extremely good, its initial versions were plagued by massive aggregation and precipitation phenomena. This effect should not be surprising, as protein concentrations in tens of milligrams per milliliter are commonplace in the stacking step. At such concentrations, the electrostatic repulsion arising from the natural electrical charge of the proteins is no longer sufficient to prevent aggregation.
The straightforward answer to this new problem is to use additives that can bind to proteins and increase their native charge. One example is the Coomassie Blue used in the BN-Page technique for separation of protein complexes (see Section 6.4 of this chapter). However, the most popular additives are charged detergents, especially SDS. SDS has the special property of binding proteins with a quasiuniform stoichiometry of 1.4 g SDS per g protein (Reynolds and Tanford, 1970). This very important binding has two favorable consequences. The first one is to make every protein strongly negatively charged. This induces a very important electrostatic repulsion and prevents almost all aggregation phenomena during elec-trophoresis. The second consequence is to mask the native electrical charge of the protein below the charges brought by SDS binding. This masking almost nullifies the influence of the native charge of the proteins and gives rise to an electrophoretic system where protein size is the sole separation parameter.
These positive features explain the very wide popularity of SDS electrophoresis as a protein separation tool. In addition to its resolution, which is quite good for a protein separation tool, since proteins differing in their mass by 2% can be separated, its popularity is bolstered by the robustness of the technique, which is applicable to almost any type of protein. The price to pay for this robustness is the denaturation of the proteins by the binding of SDS, and this technique cannot be applied to the separation of native proteins.
There are, however, a few exceptions of proteins that do not behave properly in SDS electrophoresis, most often due to poor binding of the detergent. The most classical example is glycoproteins, but very basic proteins such as histones or halo-philic proteins also show an aberrant migration via SDS electrophoresis. The other drawback of SDS electrophoresis is that it performs best at alkaline pH, where some native post-translational modifications are labile (e.g., esterification of car-boxyl groups) and where artifactual modifications (e.g., acrylamide adducts) can arise.
To counteract these problems, systems running with cationic detergents at low pH have been proposed. Although they fulfill the requirements devoted to wide-scope systems (MacFarlane, 1983), they have never achieved broad popularity. This lack of use is due to the fact that polymerization of acrylamide at low pH is difficult and uses rather erratic initiators (MacFarlane, 1983), and also because the resolution is not equivalent to the one that can be reached with SDS electrophoresis and is often not strictly correlated to molecular weight (Lopez et al., 1991). One positive side effect of cationic detergent electrophoresis is that it can be compatible with some preservation of protein activity (Akins et al., 1992). The only system that has reached some popularity in proteomics is the so-called BAC-SDS system (MacFarlane, 1989), which combines a cationic detergent electrophoresis in the first dimension with SDS electrophoresis in the second dimension. Because of the deviations from ideality mentioned above for cationic detergent electrophoresis, the separation is not diagonal but rather cone-shaped. Although the overall resolution is rather low, the system has shown its ability to separate hydrophobic membrane proteins (Hartinger et al., 1996), which are often very difficult to separate with other, higher resolution, electrophoresis systems.
Apart from these minor problems, the main drawbacks of SDS electrophoresis are its inability to separate protein forms differing by a very low mass increment (typically post-translationally modified proteins) and the difficulties encountered for the recovery of whole proteins from the gels after separation. The only really versatile system is blotting onto an adsorptive membrane, but this just replaces entrapment in a gel by adsorption on a surface and does not warrant recovery in a liquid phase. Recovery in a liquid phase, either by passive elution, or by electroelu-tion of excised bands, or by continuous elution in a buffer after electrophoresis, is often associated with variable yields and significant dilution of the proteins or is thus not widely applicable in proteomics.
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