Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
There are many ways to separate proteins from each other, based on a variety of physiochemical characteristics. These include affinity for a particular ligand, apparent molecular weight (MW), presence of ionic or hydrophobic patches and isoelectric point. Each technique has its pros and cons, and often more than one method is applied orthogonally to better characterize a target protein of interest or the specific modifications to the protein.
For querying the proteome for the presence of post-translational modifications (PTMs) like phosphorylations, nothing is better suited than the 40-plus-year-old technique of 2D gel electrophoresis (2DGE)—isoelectric focusing (IEF) followed by SDS-polyacrylamide gel electrophoresis (PAGE).
Here we review some of the different ways to examine proteins based on their charge properties.
One dimensional
There are a variety of ways to separate proteins based on their charge properties, and sometimes this is used as an end in itself or as a preparation for a technique such as mass spectroscopy (MS). It is also utilized as a first step in a 2D or even multidimensional separation. Chromatofocusing, for example—essentially ion exchange chromatography in which a pH gradient is generated, and isoelectrically focused proteins are eluted, using amphoteric buffers—can be used separately or as part of multidimensional liquid chromatography (MDLC) to separate proteins differing by as little as 0.02 pH units. Such techniques are perhaps best used as a polishing step on partially purified samples, according to GE Healthcare’s “Ion Exchange Chromatography & Chromatofocusing Principles and Methods” handbook.
The other principal method of separating proteins by charge is to allow them to migrate along a pH gradient in an electric field, coming to rest at the pH at which their net charge is zero—the protein’s isoelectric point (Pi). This is the technology behind capillary isoelectric focusing (cIEF) instrumentation. Dedicated units, such as Sciex’s PA800 Plus and ProteinSimple’s iCE line, are among the mainstays used by BioPharma to characterize and examine PTMs of their target protein in sometimes complex solutions.
Proteins can be electrophoresed on gels or strips. Ampholytes (small molecules that contain both acidic and basic groups that exist as zwitterions in specific pH ranges) are embedded in a gradient along a solid support medium, and the protein migrates to its Pi upon introduction of an electric charge. Gradients can be broad to enable separation of a wide range of Pi values—from pH 3 to 10, for example—or narrowly focused to enable a finer resolution of proteins with close Pi values, as would be found upon addition or removal of a phosphate or acetyl group.
Several vendors, including Serva, Thermo Fisher, GE Healthcare Life Sciences and Bio-Rad, offer pre-cast IEF gels of various sizes, with varying numbers of lanes and pH ranges. The gels typically are cast of acrylamide, but some, like Lonza’s IsoGel™, can be made of agarose to enable separation of larger proteins, such as antibodies. The gels may be designed to run in either vertical or horizontal (flatbed) rigs. They may, like Serva’s PreNets™, be cast with a net to allow for easy handling and electroblotting. Or, like GE’s DIGE Gel, they may be housed in a cassette that enables fluorescent imaging.
But scientists—especially in the research, as opposed to the QA/QC, world—often follow up IEF with a second-dimension SDS-PAGE separation. To do so, researchers typically run the first dimension on an immobilized pH gradient (IPG) strip rather than on a gel. That strip is “really nothing more than an IEF gel where the ampholytes have been covalently attached into the acrylamide matrix” and dried onto a plastic backing, notes Gene Stewart, president of Biophoretics. The strip separates the sample into 10, 15, 20 or more relatively distinct groups of isoforms. Upon completion, the IPG strip is set into a large single well of an SDS-PAGE gel and sealed in place with agarose, after which the samples are pulled into the gel and separated by MW as they would be in single-dimension SDS-PAGE.
Some IEF rigs, like the Serva HPE™ BlueHorizon™, can run up to 24 IPG strips and six large-format horizontal gels (IEF and SDS-PAGE gels), Stewart says. Others, like the 12,000-volt, Peltier-cooled Hoefer IEF100, run up to six IPG strips faster than any other rig, says Andrew Johnson, global product marketing manager at Harvard Biosciences. And each channel in Bio-Rad’s PROTEAN i12 isoelectric focusing tray is independently powered, enabling precise control over each of the 12 IPG strips, enabling the researcher to “survey a variety of samples, protocols and pH gradients in a single run,” says Kenneth Oh, the company’s applications and collaborations product manager for protein quantitation.
If SDS-PAGE is not in the cards following IEF, charge-separated samples can be eluted from the IPG strips. In addition, there are at least two instruments—the Agilent 3100 OFFGEL Fractionator and the Thermo Fisher Scientific ZOOM® IEF Fractionator—that utilize IPG strips but leave the isoelectrically focused samples in solution in distinct compartments. Samples can then be drawn off by pipette for downstream applications, such as MS analysis.
Sample prep
The initial sample preparation can have an enormous impact on the quality of an IEF run, as it is performed under near-native conditions. Many chaotropic reagents, ionic detergents and other denaturing agents used in protein solublization for SDS-PAGE, for example, can interfere with IEF, notes Colin Heath, director of R&D at G-Biosciences. Contaminants such as lipids and sugars that are released from the cell can affect the apparent charge (and molecular weight) of the proteins, as well.
Reagents optimized for (or at least compatible with) IEF should always be used. “We have a whole line that’s designed for 2D electrophoresis sample prep,” Heath says. These include nonionic or zwitterionic chaotropic extraction buffers, desalting agents, in-tube dialysis systems and detergent removal columns, among others.
And the addition of carrier ampholytes helps maintain protein solubility and provides maximum resolution, explains Oh.
More resolution?
While some will proceed with only the first (IEF) electrophoresis step in order to determine the isoelectric proints of the sample protein, most researchers go on to separate their proteins by a second, SDS-PAGE step, separating by molecular weight. Thus, 2DE can resolve a single protein in a mixture of thousands, and “that individual protein will show up on the same part of the gel, time after time,” notes Oh.
This is especially important when trying to visualize modified proteins. “You won’t be able to clearly see the difference between unphosphorylated, singly phosphorylated or doubly phosphorylated proteins [on single-dimension SDS-PAGE], because the MW difference is quite small—but the charge is different,” notes Joe Hirano, product manager for 2D electrophoresis at GE Healthcare Life Sciences. “You’ll start seeing a train of spots in 2DGE in the horizontal—the charge—direction from the same protein but with different phosphorylation states. You can also see the effect of charge in sugar-chain differences.”
A technical limitation of 2DGE is reproducibility. “The position of those spots is extremely important, but every gel is slightly different, and it’s really difficult to match several gels,” Hirano says. A popular fix for this has been 2D fluorescence difference gel electrophoresis (2D DIGE), in which different samples are pre-labeled with size- and charge-matched fluorescent dyes “so that it won’t affect the migration during the first or second dimension,” and run together on a single gel, with or without an internal standard. The other, older way “is to run two separate gels and use software to compare spots on the gels,” explains Johnson.
If not using DIGE, 2DGE is visualized in the same ways as single-dimension PAGE: by using either coomassie blue or silver stain.IEF gels and IPG strips also can be probed with affinity reagents. Alternatively, Protein Simple’s Peggy Sue and Sally Sue systems perform automated, higher-throughput IEF separation using nanoliter volumes separated in capillaries followed by immunodetection. These unique systems give researchers an alternative means to examine different modifications (i.e., phosphorylation, glycosylation) of proteins as long as there is an antibody to the target protein. Athough they are not 2DGE in the traditional sense, the results can be more informative.
Proven technology
The migration away from 2DGE in favor of MS by many proteomics cores, like the University at Albany-SUNY’s Proteomics and Mass Spec Facility, may be more about supporting higher-tech, expensive and difficult-to-master technology than a reflection of the merits of 2DGE. For example, Qishan Lin, who is director of the core facility, thinks 2DGE may still be the best way to separate and visualize PTMs. Tool providers will continue to improve reagents, ampholytes and separation systems to enable researchers to better characterize their proteins of interest.
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