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MIT, Broad Team Combine Enzymatic Tagging and MS for High-Res Profiling of Organelle Proteomes

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Researchers at the Massachusetts Institute of Technology and the Broad Institute have developed a method combining enzymatic tagging and mass spectrometry to enable high-resolution mapping of organelle proteomes.

The technique, which they detailed in a paper published this week in Science, offers significantly improved sensitivity, specificity, and spatial resolution compared to existing organelle proteomic approaches, Alice Ting, an MIT researcher and leader of the study, told ProteoMonitor.

Traditionally, researchers have used techniques like density gradients to isolate and purify organelles for proteomic analyses. However, Ting said, these purification processes are "highly imperfect, so you often lose a lot of material and pick up a lot of contaminants."

She and her colleagues, on the other hand, set out to biotin-label proteins in specific organelles – the mitochondrial matrix in the case of the Science paper – which would allow them to then pull out these proteins for mass spec analysis. To do this, they needed an enzyme that they could target to specific cellular regions of interest and which would biotinylate only proteins within a narrow range.

These requirements in mind, the researchers chose ascorbate peroxidase, APEX, an enzyme that they had recently engineered as a genetic tag for this purpose as well as for electron microscopy experiments.

As the authors noted, APEX oxidizes phenol derivatives to phenoxyl radicals that covalently bind with a variety of amino acids, meaning that APEX and biotin-phenol can interact to create biotin-phenoxyl radicals that will then promiscuously tag nearby proteins, enabling their subsequent enrichment.

APEX was a particularly good choice because the short half-life of these phenoxyl radicals means they have a small labeling radius – less 20 nanometers according to previous EM measurements, Ting said. That, she noted, gives the technique very high spatial resolution, which is key for investigating organelles and other small cellular compartments. By fusing APEX to a peptide targeted to their compartment of interest, the researchers were able to tag proteins highly specific to that area of the cell.

"If [the method] generated a reactive species that just spray-painted the entire cell, that wouldn't be useful," Ting said. "But it's generating a species that is only labeling proximal proteins."

She noted that in the case of the mitochondrial matrix, the enzyme's labeling radius wasn't actually put to the test because the matrix is membrane-bound. "It tested the specificity of labeling on one side of the membrane, but not the labeling radius," she said.

However, Ting said that in unpublished work she has applied the technique to non-membrane bounded components, and that the specificity appears to be high.

The method also allows researchers to zero in on regions that would be otherwise impossible to isolate for proteomic analysis, Ting said.

"For example, the intermembrane space of mitochondria – how do you purify that?" she said. "Or the synaptic cleft, or contact sites between mitochondria and the [endoplasmic reticulum]. These are impossible to purify, but the can be accessed by our method."

Such proximity labeling methods "could have great potential in cell biology and proteomics," University of Cambridge researchers Kathryn Lilley and Tony Jackson, who both work in organelle proteomics, but were not involved in the research, told ProteoMonitor this week via email.

"In principle, the method could be used to produce fine interaction maps of organelle proteins in situ," they said. "Proximity labeling could allow the analysis of specific sub-cellular and even sub-organelle niches, as long as the peroxidase can be precisely targeted."

In the Science study, Ting, who collaborated with her MIT colleague and co-author Steven Carr on the mass spec analysis, identified 495 proteins from the mitochondrial matrix. Of those, 464 proteins had previously been identified as mitochondrial, demonstrating the technique's high specificity. The remaining 31 proteins, the authors speculate, "may be newly discovered mitochondrial proteins." They tested this theory by imaging six of them, finding "complete or partial mitochondrial localization" for all six.

The researchers analyzed the proteins on a Thermo Fisher Scientific Q Exactive instrument, achieving high coverage for roughly 85 percent of the proteins. Due to the enrichment enabled by the biotin labeling, they were equally successful in detecting both high- and low-abundance proteins; however, the authors hypothesized that the technique was unable to detect some proteins that were tucked within macromolecular complexes and thus inaccessible to the phenoxyl radicals.

Ting said that given the small size of those radicals, she doubted other chemistries would have more success reaching these proteins. However, she said that "this is potentially useful information. It's a way to do kind of structural mapping of these macromolecular complexes in living cells because we may be able to biotinylate proteins on the outer shell and not biotinylate the things on the inside – and that's useful information."

In addition to their mitochondrial matrix work, the researchers have used the method to profile the proteome of the intermembrane space. That work, Ting said, has not yet been published.

Ting and her team are also developing additional reagents with the aim, she said, of building a portfolio of enzymes that can work over different distances.

"We want to have fine control over the labeling radius, to be able to shorten it and increase it depending on the application in mind," she said. "We want to be able to say, 'OK, now we want to map things that are within five nanometers; now we want to map things that are within 200 nanometers.'"

They also hope to develop chemistries for labeling additional types of amino acid side chains, she said, such as enzymes "that have more promiscuity for different functional groups."

The researchers have filed patents covering the technique, but Ting said they have no specific commercialization plans at the moment.

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