Electrophoretic prefractionation: new commercial tools from an old concept

As the field of proteomics analysis matures and is receiving wider attention from the scientific world, many new commercial tools are being marketed to meet existing needs or new work flow. Many more new types of tissues or organisms are being examined with proteomic techniques. However, even samples of lower protein complexity, such as urine and some biofluids, will require unique sample preparation or fractionation schemes. Reproducible fractionation steps will break down the protein complexity while concentrating low abundant protein species, resulting in more confident protein identifications and quantitation by 2D gels, mass spectrometry (MS), protein microarrays or other means.

Protein separation based on electrophoretic mobility of proteins is an established concept and normal practice for some years. Prefractionation of proteins based on electrokinetic methodologies in free solution, essentially relying on the isoelectric focusing (IEF) principle, has gained wide acceptance. Many commercial devices are now constructed to take advantage of this principle. Hence, consumers can be confused with the many different looking tools. To access some of these commercial products, a short review of the available technology and equipment is necessary for consumers to appreciate the many options now available to them. Traditionally, proteins can be separated by 2D gel electrophoresis, with IEF of proteins on immobiline immobilized pH gradients (IPG) strips followed by mass separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Reagent companies such as Bio-Rad (CA, USA) or GE-Amersham BioSciences (NJ, USA) have dominated the market with these low cost, yet reproducible reagents [101,102].

Bio-Rad also markets a device known as the Rotofor^® using a similar principle of electrophoretic separation in a free zone. The preparative scale Rotofor can handle up to 1 g of protein, in a total volume of 55 ml; while the mini-Rotofor can accomodate 18 ml of sample. The device has 20 sample chambers separated by liquid permeable nylon screens with cation and anion exchange compartments at its extremities. At the end of IEF with conventional carrier ampholytes, the narrow isoelectrc point (pI) range protein fractions are collected. Lubman and colleagues have directly purified the fractions with nonporous reversed phase high performance liquid chromatography (HPLC) and digested with trypsin, followed by matrix assisted laser desroption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) [1]. Likewise, Xiao and colleagues have used the device to study peptides instead of proteins, since certain peptide species have carrier ampholyte buffering ability and can generate an autofocusing pH gradient [2]. However, in general, it is more difficult for the Rotofor to generate protein fractions of consistent pI cutoff. Carrier ampholyte–generated pH gradients are labile and decay over time.

For more consistent pI separation, there are devices such as the Proteome Systems’ (MA, USA) multicompartment electrolyzer (MCE) [3,103] or Invitrogen’s (CA, USA) Zoom IEF Fractionator [104]. The MCE is compartmentalized by immobilized pH membranes that do not allow free exchange of solution between chambers. The isoelectric membranes, made of acrylic monomers, allow more precise cutoff of pI. However, precipitation of proteins on the membrane is often an issue after focusing. It is also difficult to have consistent protein recovery between runs. The fractionated proteins can be directly applied on standard narrow IPG strips for 2D gel electrophoresis, since the samples are isoelectric and isoionic. A higher sample load of defined pI range proteins on 2D gel allows the detection of lower abundant proteins. In recent years, smaller chamber and narrower pI range membranes are available. The Zoom IEF Fractionator has encompassed these features and can be viewed as a miniaturized version of the MCE.

Similar to pI separation of the MCE or the Zoom IEF Fractionator, Beckman-Coulter (CA, USA) has launched a platform (PF-2D) of protein prefractionation based on the chromatofocusing principle [105]. The platform first separates intact proteins by pI and collects upwards of 20 fractions based on pH intervals, followed by reversed-phase chromatography and MS analysis. The chromatofocusing primarily works best from pI 4–8 but the result rivals traditional 2D-electrophoresis (2DE) output, with quantitative values of protein bands particularly useful in a differential analysis of multiple samples. However, the cost for the PF-2D is significantly higher than the MCE or Zoom Fractionator. For an even more refined pI separation, one can consider the digital Proteome Chip from Protein Forest (MA, USA) [106]. The Protein Forest technology separates intact proteins by protein charge and size. Each charge separation can be precise to 0.05 or less pH units. An electric field is applied to the protein in solution until each protein finds equilibrium at its pI. Proteins from each pI fractions are then resolved according to their molecular weight by applying an electric field parallel to the polyacrylamide gel lane.

Another protein prefractionation technology is marketed by Becton Dickinson (NC, USA), utilizing the principle of free-flow electrophoresis (FFE). Originally, FFE was designed to purify cells and subcellular organelles which can recover highly purified components such as mitochondria and proteins within the proteasome complexes and can retain enzymatic activity [4,5,107]. The sample is injected into a separation chamber consisting of two parallel plates. The plates are flanked by two electrodes that generate a high-voltage electric field perpendicular to the laminar flow. Free-flow isoelectric focusing (FF-IEF) of charged particles such as proteins or cellular organelles is deflected, allowing for subsequent separation and/or fractionation. Hoffman and colleagues have successfully applied the FF-IEF followed by SDS-PAGE and electrospray ionisation mass spectroscopy (ESI-MS), on the study of cytosolic proteins of a human colon carcinoma cell line [6]. They can also recover large proteins, such as the 116 kD vinculin. Similar to the FF-IEF, other prefractionation techniques have incorporated other additional separation attributes, such as protein mobility and size, in conjunction to pI. An electrokinetic membrane apparatus, known as the Gradiflow, is such a unit from Gradipore (Frenchs Forest, Australia) [108]. It consists of three molecular weight cut-off membranes in a cartridge formation positioned between electrodes. The membranes are stacked with two parallel stream paths to form a cartridge. An electric field is applied across the membranes and streams, resulting in charged molecules transferring between streams towards the electrode of opposite charge. Molecular weight cut-off (MWCO) of membranes provides the selective means for size separations, while the pH of the buffer system allows IEF application specific protein separations.

With the many commercial tools now available to researchers, it is no wonder why every laboratory has their unique protein analysis workflow. Usually, every researcher will design a workflow that utilizes their equipment and expertise to examine the tissue of their interest. The laboratory with a bigger operating budget tends to have more options to pursue newer technology and approaches. However, buyers should be aware that all the electrophoretic prefractionation techniques have their strengths and limitations. Some approaches might be better than others in certain situations. There is no one-size-fits-all, but a rational calculation of scientific return. Clearly, prefractionation can diminish protein complexity of tissues and allow possible detection of lower abundant proteins.

Other classical prefractionation techniques such as chromatographic separation can also complement well with pI–based protein separation. Likewise, existing techniques should allow newer technology to be incorporated into its workflow. For instance, a step-wise gradient density centrifugation device from Prospect Biosystems (NJ, USA) can quickly provide reproducible protein fractionation using the sucrose gradient separation principle [109]. Extraction may start at any density step of interest without the need for going through the whole gradient and allows low-abundance protein isolation in a defined subcellular compartment. The resulting protein fractions can be analyzed with gel electrophoresis, MS or protein microarray. With the many new instruments and technologies available, we should expect a more vibrant and exciting proteomic world developing in the coming years.