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Scripps Scientists Develop Mass Spec Technique to Measure Biomarker of Oxidative Stress

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By Adam Bonislawski

Scientists at the Scripps Research Institute and the University of Michigan have developed a method for quantifying cysteine S-hydroxylation, a post-translational modification that could be useful as an early marker of oxidative stress.

The technique enables detection and measurement of protein S-hydroxylation levels via mass spectrometry, enhancing researchers' ability to investigate oxidation-reduction pathways – a form of protein signaling that could prove as important to disease research and drug development as protein phosphorylation, Kate Carroll, associate professor of chemistry at Scripps and one of the method's inventors, told ProteoMonitor.

Cysteine S-hydroxylation is "an early marker for oxidative stress, which is something that we really haven't had before," she said. "The markers people have looked at in the past are really sort of end-of-the-line, very extreme markers of oxidative stress – i.e., the cell is dying. The earlier the indication we can have that oxidative stress is going on, the better we can profile it and potentially even predict or profile changes [related to] disease."

The technique should also prove helpful in unraveling the basic structure of redox signaling pathways, Carroll added, noting that very little work has been done in that area due to the lack of technologies for approaching it.

"There's been virtually no work done to map these signaling pathways even though we know these modifications are very important because we simply haven't had the tools," she said. "Redox signaling is critical for many different cell types, so we'd like to use the technology we've developed to map these signaling pathways the same way people have used [similar] technologies to look at phosphorylation."

The method, which is described in a paper published this month in the online edition of Angewandte Chemie, uses isotope-coded dimedone and iododimedone to tag the sulfenic acid that results from cysteine S-hydroxylation. This allows the modified cysteine sites and their proteins to then be identified using mass spec.

Carroll first identified sulfenic acid as a biomarker for cysteine S-hydroxylation in a 2009 study in which she tagged the acid with a fluorescent antibody, enabling her to look at oxidation levels inside cells. The new technique builds on that approach by allowing the quantification of sulfenic acid levels on individual proteins.

Carroll said she plans to commercialize the assay as a kit through Ann Arbor, Mich.-based Cayman Chemical, which also offers a kit based on the 2009 assay. Additionally, her lab has developed an antibody against the covalent bond created when the isotope-coded dimedone reacts to the sulfenic acid. Sold by Millipore, the antibody is intended for researchers looking to enrich for cysteine S-hydroxylation-modified proteins prior to mass spec analysis.

Carroll "has been at the forefront of developing chemical methods for qualitatively assessing protein sulfenic acid modifications," Matthew Wood, an assistant professor in the department of Environmental Toxicology at the University of California, Davis, told ProteoMonitor. "This is really an extension of that work. It's an ingenious way of quantitating protein sulfenic acid."

Carroll's lab developed the method on a Thermo Fisher LTQ ion trap instrument, she said, switching to an Orbitrap machine on occasions where high sensitivity was needed. While she likened the study of redox pathways to the investigation of protein phosphorylation, the mass spec work involved in redox studies will probably be more straightforward, she suggested.

Identification of phosphopeptides by mass spec remains "an emerging area for two reasons," she said. "One, people are still working out good strategies to enrich specifically for phosphorylated peptides. Two, you have to use a very specific kind of ionization, otherwise you lose what is a very labile kind of modification."

That's not the case with sulfenic acid modification, she noted. "Once we trap [the sulfenic acid modification] with the reagents we've developed, it's a stable modification and we don't need to go to great lengths to preserve some kind of labile modification."

Her lab is also using the technique to localize the specific cysteines receiving the modification – something that has traditionally proven a difficulty in mass spec-based PTM work (PM 11/12/2010).

"I think that's key to understanding" redox signaling, she said. "It's an important aspect of the research to be able to look at different cysteines within a protein, to address why some cysteines are prone to oxidation and this switch-like behavior and others are not."

As with other PTMs, key to such localization efforts is getting good digestion of the protein being investigated, Carroll said, noting that "for the proteins we’ve been looking at we can get 75 to 80 percent coverage."

The most significant difficulty her team has run into using the technique has been the lack of software specific to analysis of sulfenic acid modifications.

"Ultimately the bottleneck isn't the mass spec or the chemical approach, but the data analysis," she said. "This isn't just an issue with our area of redox signaling. Anything that's really a non-standard modification, you have to work with IT folks to work around it. The Mascot program that everyone uses just isn't designed for modifications that are non-standard, so it's just a general problem in the proteomics field – looking at any modifications that aren't, quite frankly, phosphorylation."

Given the interest in the relation of oxidative stress to various diseases, Carroll said she expects the technique will draw attention from drug developers. She has received inquiries from several pharmaceutical and biotech companies, she noted, although she didn't name any firms specifically.

"I'm on the advisory board of a number of [pharma] companies that hope to exploit" the technique, she said. "Any kind of change in the redox balance within a cell can mean either disease or the initiation of disease or the progression of disease. So there is a ton of interest in being able to manipulate the redox state of the cell."

"One application of the technology is the identification of new targets we can use to modulate a particular disease," she added. "Another is monitoring the utility of reagents people have designed to modulate redox homeostasis to see whether the small molecules that have been developed [to modulate redox levels] are actually working or not."

The extent to which redox modifications affect disease states "has been a largely unexplored area," UC Davis' Wood said. "Whether blocking sulfenic acid modification with some sort of drug can prevent disease remains to be seen. It potentially could down the road."

"What you'd like to be able to do is associate a redox modification with some sort of biological outcome, and a key component to assessing whether the modification is biologically relevant to a cell is being able to quantitate the amount of modification that's occurring," he said.

Going forward, Carroll plans to continue work on the assay itself – in particular on developing improved capture agents and a fluorescent probe that could be used for imaging – while applying it to the study of various signaling pathways and diseases.

"I would say the number one disease is cancer," she said regarding the technique's potential research applications. "Again and again cancer is associated with oxidative stress. There's also been a lot of work in the area of neurodegeneration and cardiovascular health. Those are three key areas."


Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

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