The use of classical electrophoretic methods has been hindered by the limited loading capacity, but many improvements have been made, due to new instrumental developments. Most electrophoretic methods are based on isoelectric focusing (IEF) separation. Below is a non-exhaustive review of IEF-based methods only.
ROTOFOR
It is in 1998 that Bier, who had long been working on preparative electrophoretic separations in free zone, developed the concept of the Rotofor (rotationally stabilized focusing apparatus), based on recycling carrier ampholytes IEF.81 The device is today commercialized by BioRad. A typical instrumental setup is presented in Figure 10.
Figure 10: Schematic presentation of the Rotofor instrument. Rotation and the screen partitioning are essential
for good separations. Reprinted from81
The apparatus is assembled from 20 sample chambers, separated by liquid-permeable nylon screens, except at the extremities, where cation- and anion-exchange membranes are placed against the anodic and cathodic compartments, respectively, to prevent diffusion within the sample chambers of undesired electrodic products. The whole setup is rotated along the axis perpendicular to the chambers, thus avoiding decantation. The initial purpose of the Rotofor was for preparative use, with a loading capability of up to 1 g of protein in a total volume of up to 55 mL. A mini-Rotofor, with a reduced volume of 18 mL is also available, and recently a micro-Rotofor sold by Bio-Rad as well.82
The resulting pI fractions can then further be used for analysis on a conventional 2D gel electrophoresis.83 But IEF with the Rotofor can also be integrated as a first dimension in a 2D methodology.43 The fractions are further analyzed by RP-HPLC in a second dimension, each LC peak is then collected and tryptically digested, before being subjected to MALDI-MS analysis. This method was successfully applied to many challenging biological protein mixtures.43, 84, 85 The pI accuracy of this method was estimated to range from ± 0.65 to ± 1.73 pI units. More recently, Xiao et al. reported the application of the Rotofor for the fractionation of tryptic peptides from human serum in an ampholyte-free environment, and showed an “autofocusing” effect.86
CONTINUOUS FREE FLOW ELECTROPHORESIS (FFE)
Figure 11: Schematic presentation of the Free Flow electrophoresis setup (commercial name Octopus).
Separation chamber dimensions are 50×10×0.4 cm (50 cm electrode length, 10 cm between electrodes and 0.4 cm chamber depth). Focused protein samples are collected into 96-well plates via an in-line multichannel outlet. The volume of each fraction is typically ~2 mL. Reprinted from87
This liquid-based IEF technique was described in 1982 by Hannig88 and more recently reviewed by Bocek et al.89 A commercial version exists under the name of Octopus.90 In FFE, the sample is injected continuously into a carrier ampholyte solution flowing as a thin film (0.4 cm thick) between two parallel plates and, by introducing an electric field perpendicular to the flow direction, proteins are separated by IEF according to their different pI values and finally collected into up to 96 fractions (Figure 11). Two main advantages of this method are the recovery of liquid fractions, and the sample loading capacity due to continuous sample feeding. FFE was used as prefractionation tool before 2D-GE,91 or integrated as a first dimension in a 2D strategy.92, 93 Conventional FFE was initially developed as a preparative- scale technique for isolation and purification purposes, but further developments have led to micro-fabricated devices (mFFE or µFFE), reported by Kobayashi et al.,94 and Manz and co- workers.95-97
MULTICOMPARTMENT ELECTROLYZERS WITH ISOELECTRIC MEMBRANES
Another apparatus that has also proved its efficiency is the multicompartment electrolyzers (MCE) designed by Righetti et al.98, 99 The device is constituted of multiple compartments, separated by a polyacrylamide gel membrane with a specific pH produced by immobilines that are incorporated into the polyacrylamide membranes (Figure 12). Thus, the principle is to capture proteins in an isoelectric trap formed by two Immobiline membranes having pI values encompassing the pI of the protein under analysis.
Figure 12: Schematic presentation of the multicompartment electrolyzers. The upper right panel shows a 2D
map of unfractionated sample vs. three different 2D maps (lower panel) of three isoelectric fractions, captured into traps having membranes with pIs 3-5, 5-6 and 6-10.5. Reprinted from100.
A commercial apparatus, called IsoPrime, incorporating this principle has been marketed (Amersham Biosciences, Piscataway, NJ, USA). The commercial unit has been developed primarily for large scale purification (about 30 mL). The device was later miniaturized for proteomics purpose101, 102 and could detect low abundance proteins unseen until then. Good results have also been obtained by Zuo et al. with the same type of apparatus being used as prefractionation tool.103, 104
While most of these devices can provide reasonable to high quality separations, the limitations encountered with either the Rotofor or the IsoPrime are the following: (1) they require a large sample volume; (2) they produce large volume, dilute fractions that need to be concentrated with attendant losses.
OFFGEL
Contrary to the previously mentioned devices, the OFFGEL was designed for analytical to semi-preparative purposes, the volumes required are much smaller compared to other techniques. However, just like the MCE, OFFGEL has been devised for IEF separation with direct recovery in solution, and without adding ampholytes to form the pH gradient.105
The principle is to place a sample in a liquid chamber positioned on top of an IPG gel. The gel buffers a thin layer of the solution in the liquid chamber and the proteins are charged according to their pI values and to the pH imposed by the gel. Theoretical calculations have shown that the protonation of an ampholyte occurs in the thin layer of solvation close to the IPG gel/solution interface.106 Upon application of a voltage gradient perpendicularly to the liquid chamber, the electric field penetrates into the channel and moves all charged species (those having pI above and below the pI of the IPG gel under the chamber) out of the chamber. After separation, only the globally neutral species (pI = pH of the IPG gel) remain in solution. This technique offers high separation efficiency and allows easy recovery of the purified compounds directly in the liquid phase. In further developments, the OFFGEL electrophoresis format was adapted to a multicompartment device (Figure 13), composed of a series of chambers of small volumes (100-300 µL).107
The resolution thus depends on the pH gradient of the underlying IPG gel, and of the number of compartments for recovery. A resolution of 0.1 pH units could be obtained when operating in narrow ranges, for example the separation of β-lactoglobulin A and B. The capability of the multicompartment device to fractionate complex biological mixture was also demonstrated, by the fractionation of an E. coli cell extract. Further developments were done and this device is now being commercialized by Agilent Technologies since last year.
The main concern about electrophoretic methods is to design instruments that effectively dissipate Joule heat or to limit that heating. In the Rotofor and multicompartment electrolyzers for example, there is a cooling system that allows temperature control during electrophoresis. For the commercial OFFGEL, the electrophoresis is performed on a cooling plate, allowing that control. But another way to limit this Joule heating is to control the maximum current/power allowed. For example, this is done in the OFFGEL device, by limiting the current to few hundreds of micro-amperes.
A summary of electrophoretic methods is given in Table 2.
Table 2: Summary of electrophoretic methods and the volumes needed.
Electrophoretic methods Usual volumes loaded Use*
Rotofor: - preparative, mini 55 mL, 18 mL P, SP
- micro 2.5 mL (ref.82) A
Free Flow Electrophoresis (FFE) - continuous FFE
Chamber: 50 (width)×10 (length)×0.4 (depth) cm
2 mL/fraction
P
- micro devices mFFE and µFFE 300 µL (ref.94) and 0.2 µL (ref.96) respectively A
Multicompartment Electrolyzers - miniaturized
30 mL up to 125 mL (IsoPrime device) (ref.103)
500 µL/chamber × 3 chambers = 1.5 mL (ref.103)
P A
OFFGEL 100–300 µL/chamber
× 10–20 chambers = 1–6 mL
SP, A