By Laura Lane
Making optimal use of precious resources is not the exclusive domain of environmentalists. You could say that some protein scientists are on the same mission. Using two-dimensional polyacrylamide gel electrophoresis (2DGE), these researchers are trying to make the most of the very narrow pipeline of viable hearts for transplantation. The task involves finding the proteins involved in transplant rejection. Accomplishing this hinges on 2DGE, a technique for separating complex mixtures of proteins. Though part of the life science toolbox for decades, 2DGE remains the method of choice for studying proteins of all kinds of physiological phenomena.
“The ‘death’ of 2DGE has been forecast for many, many years, as it is considered a dated technology,” says Bob Marchmont, global marketing director for GE Healthcare’s 2-D labeling and detection group. “And, yet, it still continues to expand and grow.”
It all began 80 years ago when an electric field was harnessed to influence serum proteins to move through various matrices, which have included paper, starch, agar, capillaries, and polyacrylamide gel. Called electrophoresis, the technique continued to evolve over the decades. The development of isoelectric focusing, which is now known as the first-dimension of separation, moves proteins through a pH gradient to a position at which they assume a neutral charge, or their isoelectric point. This is achieved with immobilized pH gradients (IPG), created by covalent attachment of buffering compounds along the length of a polyacrylamide gel. Cast on to a plastic backing and later cut into strips, the IPGs can easily be handled and manipulated.
The second dimension of separation is best known as the acronym SDS-PAGE. Spelled out as sodium dodecyl sulfate-polyacrylamide gel, the procedure uses electrophoresis to influence proteins to move according to their molecular weights. First, IPG strips, containing focused proteins, are treated with SDS, which eliminates secondary and tertiary protein structures that could affect the proteins' mobility. This ensures that molecular weight is the sole variable for differential migration patterns. In addition, SDS introduces a negative charge to the proteins. Reducing agents may also be used to breakup structures that involve disulfide bonds, which are then blocked with iodoacetamide to prevent reformation of the disulfide bonds. The IPG strip is then placed on the SDS gel for electrophoresis.
“Although it’s not generally considered ‘cutting-edge’ technology, in reality, 2DGE still remains the best of all available protein separation approaches,” Marchmont says. “It’s a relatively low investment technology, familiar, and provides levels of information that other technologies cannot.”
That may include the landscape of protein expression during the rejection of a transplanted heart. Using 2DGE to examine proteins of biopsy samples of transplanted hearts, researchers pinpointed 100 proteins for which the expression levels increased by two- to 50-fold during rejection. Further study revealed that two of the proteins were also present at elevated concentrations in blood serum. [1] The identification of these proteins could open the way for detecting rejection through blood samples instead of more costly and invasive biopsies.
Finding such biomarkers fuels much of the enthusiasm for 2DGE. “It’s the only technique that gives you the ability to look at thousands of proteins at the same time,” says Bill Gette, marketing manager for electrophoresis in the laboratory separations division of Bio-Rad Inc.
By staining the proteins after electrophoresis or labeling proteins with dyes beforehand, 2DGE results in a constellation-like image that can be digitally scanned and analyzed with software. The market offers a variety of digital imagers, ranging from charge-coupled device (CCD) cameras to laser-based scanners. You can purchase stand-alone image analysis software or use packages that come with the imaging device. While visual inspection is sometimes sufficient, you’ll probably need the help of software if you’re separating thousands of proteins all at once. Analysis programs also generate quantitative information on expression levels, provide the sensitivity to detect faint spots, and filter out background noise.
“Software is a critical component,” Marchmont says. “It brings to biologists sophisticated analysis and statistical tools, but in a way that biologists can answer questions that they want to ask of the data.”
Protocols have also evolved to improve analysis. The development of difference gel electrophoresis (DIGE) provides a solution to the inherent variabilities of 2DGE, reduces the “experimental variation and allows scientists to accurately and reproducibly focus on the true biological variation that is of interest,” says Marchmont, who credits GE Healthcare for introducing this second-generation gel separation paradigm. The procedure calls for labeling two protein mixtures with two different cyanine dyes. A separate pool of all the samples to be compared is also labeled with a third dye and an aliquot included with each pair of samples. All three are separated on the same gel. “The size- and charge-matched dyes allow for co-migration of identical proteins,” he explains, allowing, for example, comparison between two patient samples.
“DIGE has taken 2DGE to a new level,” says Marchmont, pointing to the more than 450 peer-reviewed published papers that mention the technique. “There’s a new publication quoting DIGE every other day.”
The company will be hosting a free webinar on May 9th at 8:00 am, and again at 2:00 pm, Eastern Daylight Time. To register, visit www.gelifesciences.com/pr-digewebinar.
Even without DIGE, 2DGE has few competitors. The biggest competitor, if any, is liquid chromatography (LC) followed by mass spectrometry (MS). However, the complexity and expense of LC/MS instruments can pose insurmountable barriers. Though they provide the opportunity for automation and high-throughput speed, LC/MS generates “raw lists of proteins present in a sample, with no information on the relative quantitative abundance of the individual protein components and give no indication as to whether the protein identified are subject to PTM [post-translational modifications],” according to a recent review published in Circulation Research [2].
With 2DGE, you can spot PTMs by looking at the position of proteins. Changes in the location of the protein spot could reflect altered isoelectric points or molecular weights brought on by phosphorylation, glycosylation, hydroxylation, and other PTMs. You can also use dyes or stains that exclusively bind to certain PTMs, such as Pro-Q Diamond that exclusively stains phosphoproteins, and Pro-Q Emerald that stains glycoproteins. Both were developed by Invitrogen Corp.
Such advances in reagents as well as for other tools have certainly expanded the range that researchers can now explore. However, companies perhaps provide the most valuable tool with their offering of information and training. For example, Bio-Rad’s 2-D doctor helps researchers with troubleshooting as an online tool or downloadable software. Users can view how-to videos and upload gel images for individual evaluation by the company’s 2DGE specialists.
Invitrogen has teamed up with the Human Proteome Organization to provide training. “If people don’t have the confidence to perform an application, then it impacts everyone,” says Evangeline Gonzalez, director of proteomics at Invitrogen. “Training helps people to run experiments properly so they can compare results with other researchers and the entire field can move forward.”
References:
[1] Borozdenkova S et al., “Use of Proteomics to Discover Novel Markers of Cardiac Allograft Rejection,” Journal of Proteome Research, 3(2):282 -288, April 2004.
[2] McGregor E and Dunn MJ, “Proteomics of the Heart: Unraveling Disease,” Circulation Research, 98(3):309-321, February 17, 2006.
