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Gel Electrophoresis Developed for Glycation Detection; Other PTMs in the Works

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By Tony Fong

Bucking current trends, researchers in the UK have developed a gel-based technology for the detection of glycated proteins that they are currently looking to develop for use with other post-translational modifications.

While the technology is not meant to replace mass spectrometry-based methods of PTM analysis, its developers said that it does offer a quicker, easier, and cheaper method to visualize modified proteins. Further, they added, the technology may eventually be used to develop diagnostic tools for diseases such as Alzheimer's, diabetes, and cancer.

The technology and its applications are described in a proof-of-concept study published Nov. 26 in the online version of Proteomics.

PTMs have become one of the leading areas of research in proteomics with a fair amount of work directed at developing new methods for delving into post-translationally modified proteins. Most of those methods under development are mass spec-based.

According to a co-author of the study, though, the gel-based technology was developed after mass specs failed for this purpose. Tony James, a reader in chemistry at the University of Bath, said that he and his colleagues had set out originally to try to separate sugars.

They first tried mass spectrometry, but it "wasn't able to detect what we were looking for," he told ProteoMonitor. They then turned to gel electrophoresis and in their analysis noticed that in addition to carbohydrates, they saw "a big band separation when [we] got a protein on our gel, as well," James said.

Jean van den Elsen, a co-author on the study and a lecturer in biochemistry at the University of Bath, said that they saw "such good results with the carbohydrates, we thought, 'Let's now look at glycosylated proteins.' And we attached a range of glycosylated proteins on the gel, and none of them were retarded by the electrophoresis."

When they looked at glycated proteins, though, differences were observed.

"We started working on a recombinant protein, which we know has a problem that is in the cells in which we produce this protein, E. coli," he said. E. coli adds a glycation to this protein, "and we were able to pick this up very quickly," van den Elsen added. "So we started looking at other proteins," including albumin "and [when] we in vitro glycated these proteins, we could actually see a big difference in their movement in the gel."

Eventually, he and his colleagues were able to detect the glycated proteins using an LC-MS platform "but it wasn't easy, whereas the gel electrophoresis showed the bands straight away," van den Elsen said.

The new technology is based on the traditional polyacrylamide gel method with one key difference: the addition of methylacrylamido phenyboronic acid, which interacts with sugar. This allowed the gel they developed to pick out glycated proteins from unmodified ones.

The boronic acid binds to the carbohydrate and "retards or slows down the receptor protein that contains the glycated unit, so it gives them an artificial mass, so they appear a lot heavier" than they should, James said.

Not all sugars are bad, however, and rather than picking out all sugars, the gel created by the researchers separates only the "bad" ones — those that have been implicated in disease.

During glycation, James and his colleagues said in their study, early- and long-term glycation products are formed. The long-term products, called advanced glycation end products, or AGEs, consist of "a broad range of heterogeneous fluorescent and yellow-brown products, including nitrogen-containing and oxygen-containing heterocycles, resulting from subsequent oxidation, dehydration, cyclization, and condensation reactions with other reactive amino groups."

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AGEs, they added "are important long-term biomarkers for ageing and age-related chronic disease states such as diabetes, cardiovascular diseases, Alzheimer's disease, autoimmune disease, and cancer."

Gel vs. Mass Spec

PTM work is dominated by mass spec-based techniques, but van den Elsen noted that methods based on that platform are not always convenient. Especially in a diagnostic setting, mass specs "are not always available," he said.

In contrast, their gel technology, which the University of Bath holds the patent to, is "very handy [and] cheap, and can be used in any lab because you don't have to have mass spec equipment to pick these things up," van den Elsen said.

Gels, however, have poorer coverage than mass specs. The researchers have not tested their technology against mass specs to compare each method's depth of coverage, but according to van den Elsen, in work they've done with whole human serum, "we can actually pick up quite a few bands of proteins that are glycated."

He acknowledged that in whole-proteome research, the technology might be "more limited" than mass spectrometry, but "I think that in some specific cases, it's a very good and better visual technique to pick up glycation … and identify different types of glycation — which sugars are attached to the protein."

The researchers are beginning to examine clinical samples with their technology. If successful, they may identify clinical uses for the approach, though in the current stage its primary use is for pure research.

Though the technology is currently in a gel electrophoresis format, it can be moved to a capillary electrophoresis system to make it more user-friendly, van den Elsen and James said. They added that it could be translated into a chip-based format to be used in a hand-held device for the detection of disease biomarkers.

In particular, they pointed to the need for a blood-based test for Alzheimer's. No such test currently exists, and "if we can develop this technique into a test, doctors could potentially diagnose patients at an early stage before their symptoms show up in a brain scan," James said.

He and his collaborators also are interested in investigating amyloid peptides that become glycated, which are "very important in research" for dementia and prion disease, van den Elsen added.

The technology may have applications for therapeutics development, also. "It's very important in the pharmaceutical industry to make sure that the recombinant proteins that they produce as biologics are free of glycation," van den Elsen said. "And this is a very quick and easy test [to see] whether your proteins are glycated and which fraction is glycated."

The technology is currently in a 1D gel format, but the researchers are working to incorporate boronic acid into a 2D gel to add glycation detection as an additional dimension.

They also are investigating different types of boronic acids in order to make the technology selective for specific types of PTMs. In their paper, the authors noted that boronate used in the study was specific for the detection of glycation and did not result in "significant interference" in other PTMs.

"At the moment we've used a simple receptor, which is the boronic acid, but you can obviously enhance its selectivity and design better receptors that are more targeted and specific," van den Elsen said, identifying phosphorylation and different types of glycosylation as potential PTMs of interest.

He said that preliminary work suggests the utility of their approach but declined to elaborate.

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