A team from the Scripps Research Institute's Florida campus has developed a chemical probe, rhodamine-phenylglyoxal or Rh-PG, to better identify citrullinated proteins as potential biomarkers in a number of diseases.
The group, from the lab of Paul Thompson, recently published a study in the Journal of the American Chemical Society demonstrating that Rh-PG, which fluorescently tags citrullinated proteins, can identify specific citrullinated biomarkers to predict and diagnose diseases like ulcerative colitis and rheumatoid arthritis.
Researchers have previously observed that these diseases are marked by high levels of protein arginine deiminases, or PADs, which catalyze protein citrullination. But, according to the Scripps team, a method affordable and accurate enough to identify specific citrullinated biomarkers was not previously available.
Kevin Bicker, the study's lead author, told ProteoMonitor this week that the team is currently talking with potential commercial partners to make the Rh-PG probes available to researchers hoping to better understand the role of citrullination in these diseases and identify diagnostic biomarker signatures.
"We've done a lot of collaborations with people who are looking at the PADs in diseases," Bicker said. "As we worked with these collaborators, they found that PADs were implicated in a lot of diseases … We knew the PADs were there, we just didn't know what they were working on. So we set out to develop a tool — our probe — that we could use to selectively label citrulline so we could actually find these disease biomarkers."
In the study, Bicker and his colleagues first showed that their RH-PG probe detection method performed as well or better than two existing methods: the color development reagent, or COLDER, assay, and a commercially available Millipore detection kit based on western blotting.
The researchers then demonstrated that they could identify specific disease-associated citrullinated biomarkers in a mouse model of ulcerative colitis using Rh-PG probes.
According to the group's paper, the Rh-PG probe design is based on a chemoselective reaction that occurs between glyoxals and either citrulline under acidic conditions, or arginine under basic conditions.
Under acidic conditions, the group found that the probe reacted selectively with citrulline, homocitrulline, and cysteine. A neutralization step in the group's gel-based screening method takes care of this cysteine cross-reactivity, the authors wrote.
Testing against the Millipore kit, the Scripps group found that Rh-PG labeling took "significantly less time," required fewer steps, and involved simpler analysis.
With the kit, Bicker said, "the biggest issue we had was [that] it's really quite expensive, $40 to $50 per assay. [Also,] the entire procedure takes about two days and requires western blotting, which is a commonly used, but less easy technique."
Regarding the COLDER assay, the problem "is really sensitivity," he said. "You can't analyze anything below about 10 to 20 micromolar citrulline, which means when you are working with really precious proteins, you just can't do that type of analysis. It becomes too expensive, too burdensome."
Bicker said that the team also found that Rh-PG labeling offered a higher limit of detection.
"With the probe we are achieving higher levels of detection, which is a big advantage when you are looking for disease biomarkers that perhaps aren’t [as] prevalent in a serum sample or cell extracts. It's good to have this sensitivity so we can pick up each marker," he said.
To see whether the probe method could detect disease-associated biomarkers, the group measured changes in protein citrullination using serum samples from a previous study that demonstrated that a PAD inhibitor called Cl-amidine reduced disease severity in a mouse model of ulcerative colitis.
"We wanted to take samples we'd looked at before and see [first] if we could confirm the results we got with the COLDER assay, but then also build on that to show that we could also look at individual proteins and look at the citrullination levels," Bicker said.
The group identified five protein bands that showed a marked change in citrullination in response to the inhibitor. Then the researchers compared florescence intensity for the five proteins to each of five different disease metrics: colon length, weight difference, white blood cell count, lymphocyte count, and inflammation score.
Colon length and inflammation scores showed the "highest number of correlations" between disease severity and citrulline level, the authors wrote. These two end points are "key indicators of disease severity," they added.
Strong correlation between citrullination of one of the protein bands and common disease activity scores suggests that it may be a biomarker of ulcerative colitis, the group wrote.
Bicker said that he and the group have gone on to do proteomics work to identify the proteins implicated in the study, but said he could not yet discuss what they have found.
"We have been able to identify some, and [have done] some characterization just to confirm they are PAD substrates," he said. "Now [we are] moving forward to see what the role of these biomarkers is in the disease."
While Bicker said the lab isn't yet working on developing the Rh-PG probe method for use as a diagnostic, it is potentially on the agenda.
Right now, "we are more interested in getting this out as a tool so our collaborators can use this to really find insights into these diseases. Once we start identifying more markers for all these diseases we may start to make a push toward converting this to a diagnostic of sorts," he explained.
While the researchers argue in the paper that the probe method offers a cheaper and simpler alternative to other tools, Bicker said the team has not yet done a cost analysis to establish exactly how much the method costs.
However, he said, "the amount of material you use per assay, if we were compared directly with the western blotting technique – we use such a small amount of material and we can make it in large batches."
"And because we synthesize it in a flask, as opposed to an antibody which you have to make in an animal – we can make much larger quantity at a much lower cost," he said.
According to Bicker, after filing a patent on the method, the group is now in discussions with a company, which he declined to name, interested in developing the Rh-PG probe method as a commercial kit.