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Japanese Team Develops Method to Tag Proteins Selectively Based on Chemical Interactions for Mass Spec ID

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NEW YORK (GenomeWeb) – A new protein-labeling method provides scientists the ability to find proteins involved in specific biochemical processes. By choosing labeling reagents that act only under particular chemical circumstances, scientists from Japan's Kyoto University said their results are a step towards conditional proteomics.

Led by first author Takayuki Miki and senior author Itaru Hamachi, the researchers developed reagents to label proteins involved in how cells interact with the chemical element zinc. The researchers used the reagents in a proof-of-concept study to show that they could use high-resolution mass spectrometry to identify proteins that were previously unknown to participate in zinc-related biology.

The scientists published their results last week in Nature Methods, showing that they could conditionally label proteins in living cells and provide information on spatiotemporal changes in the zinc-related proteome.

"To the best of our knowledge, this is the first report on a condition-focused proteome analysis," the authors wrote. "By applying Zn2+-responsive chemical tagging, our method can take a snapshot of dynamically altered conditions that are readily destroyed by a lysis process in a conventional proteomics analysis."

While the method depends on finding and applying appropriate labeling reagents, there are several chemical conditions to which the method might be easily extended.

"Although we focused on the zinc-associated signaling here, we envision that this conditional proteomics strategy can be extended to other conditions such as other metals, pH, hypoxia/hyperoxia, or particular enzyme-enriched conditions," the authors said, naming copper and iron ions in particular.

"Synthesis of our labeling reagents for zinc is quite easy," Hamachi told GenomeWeb in an email. Already, the lab has begun working on several other kinds of reagents with different affinities for zinc. Each chemical "condition" will need specially designed and validated reagents, "but the design guideline shown here can be used for other metals," he said.

The authors suggested that other groups could follow their study as a guideline for designing other labeling reagents. "Many fluorescent chemosensors have already been reported according to the same design strategy," the authors wrote. "In addition, caging groups that mask the original activities and release the active species upon stimuli may suggest an alternative for designing probes responsive to other conditions. These efforts should enrich useful platforms to elucidate many biological processes associated with a variety of conditional changes."

Hamachi's lab features several groups, including one working on protein detection and another on chemistry in neurobiology. He said that his lab became interested in finding proteins involved in zinc biochemistry because it is a key metal in neuron and brain function, especially in brain disorders.

Zinc bound to proteins serves structural and catalytic functions and free zinc potentially serves as a signaling molecule. Studies suggest that the distribution of zinc changes over space and time, and while fluorescent probes enable real-time imaging of zinc ions, they "cannot characterize new proteins that could be indirectly yet significantly associated with zinc distribution, for example, constituent proteins of zinc-rich vesicles," the authors wrote.

And while some techniques, like ascorbic peroxidase reporters, can label proteins on a spatially-restricted basis, the authors said that there hadn't been a way to restrict labeling based on desired conditions, like the presence of zinc.

To elucidate the zinc-related proteome, the scientists designed reagents to label proteins only in the presence of high concentrations of zinc ions. Called "AIZin," the labeling reagent features a Zn2+-binding site as well as a reactive moiety, in this case dipicolylamine and acyl imidazole, respectively.

The scientists developed two slightly different AIZin varieties and showed how they used one, AIZin-2, to label proteins in live cells where nitric oxide triggered a rise in levels of intracellular zinc ions.

Furthermore, using mass spec analysis, the scientists were able to identify 72 proteins involved in zinc biology including seven that had previously been associated with zinc ions: calreticulin (CALR, top 1/331), protein disulfide-isomerase A3 (PDIA3, 2/331), peptidyl-prolyl cis-trans isomerase A (PPIA, 5/331), endoplasmin (ENPL, 8/331), elongation factor 1 alpha 1 (EF1A1, 13/331), ADP-ribosylation factor 4 (ARF4, 15/331), and Ras-related protein Rab1A.

Hamachi said that high-resolution mass spectrometry was essential for identifying new proteins. For this study, his team used nanoflow liquid chromatography followed by tandem mass spec on a Thermo Fisher Scientific LTQ-Oribitrap XL instrument.

Hamachi pointed out that the speed at which the reagents operate would provide some limitations. In environments where proteins or zinc ions are diffuse, such as in intercellular space, the method might not work so well. "We do not think that direct interactions are necessary, but rather it is crucial for reagents and targets to stay together in a closed space sufficient for labeling reactions," he said.

He added that his lab plans to use the newly discovered proteins as targets for selective imaging or monitoring in live cells and in other cell types, and that the method could be applied to evaluate drug candidates' effect at the proteomic level.

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