NEW YORK (GenomeWeb News) – A new approach is using an engineered peroxidase enzyme called ascorbate peroxidase, or APEX, to help label and catalog proteins within specific compartments of living cells. Massachusetts Institute of Technology, Harvard Medical School, and Broad Institute researchers presented the method in a study appearing online yesterday in Science.
The researchers demonstrated that they could tally up nearly 500 proteins present in human mitochondria using genetic techniques to target APEX to the organelle's matrix-facing inner membrane. Within that compartment, the enzyme specifically tagged neighboring proteins through a process called biotinylation, they explained, making it possible to isolate and identify the proteins by conventional methods.
In the mitochondria, for instance, the approach proved useful for pinning proteins to the organelle that had not been found there in the past, while accurately distinguishing between proteins facing into or out of the mitochondrial inner membrane.
Those involved in the study said the same strategy should work for doing proteomic analyses in other cellular compartments as well.
"The specificity of live-cell peroxidase-mediated proteomic mapping combined with its ease of use offer biologists a powerful tool for understanding the molecular composition of living cells," senior author Alice Ting, a chemistry researcher affiliated with MIT and the Broad, and her colleagues said in the paper.
Some of the most commonly used proteomic approaches involve microscopic assessments of a few selected proteins marked by tags such as the green fluorescent protein in living cells, researchers explained, or mass spectrometry-based techniques that require careful purification steps to find proteins within a specific organelle in the cell.
With their new APEX-based strategy, though, Ting and her co-authors argued that they have come up with a method that brings together the benefits of both microscopy and mass spec, "offering spatially- and temporally-resolved proteomic maps of endogenous proteins within living cells."
"Our approach was to tag the proteome of interest with a chemical handle such as biotin while the cell was still alive, with all membranes, complexes and spatial relationships preserved," they wrote. "We thus required a genetically targetable labeling enzyme that covalently tags its neighbors, but not more distant proteins, in living cells."
To that end, the team settled on a system hinging around APEX, an engineered enzyme that they also explored as a genetic tag in electron microscopy.
Because APEX can turn the chemical phenol into phenoxyl radicals that react with certain amino acid residues in proteins, the investigators suspected that the enzyme might come in handy for proteomic studies, too.
In their new proof-of-principle study, the researchers directed APEX to the inner mitochondrial membrane of a human embryonic kidney line. Once there, they prompted the peroxidase enzyme to label its protein neighbors with biotin by adding hydrogen peroxide as well as biotin-phenol.
After letting biotinylation take place for a minute, the group purified the tagged proteins with streptavidin-coated beads capable of nabbing the biotin tags on the proteins. From there, these purified protein samples were assessed using mass spec.
In the two cracks they took at this analysis, researchers unearthed 495 mitochondrial matrix proteins from the human cells. Among them were 31 proteins not found in the mitochondria in the past, the study's authors noted, along with 240 known mitochondrial proteins whose precise location within the mitochondria had not been previously described.
Half a dozen other proteins placed in the inner mitochondrial membrane in the new analysis had been linked to alternative sites in the mitochondria in prior studies. Even so, at least five of the six proteins showed up at the inner membrane site tested in follow-up experiments, supporting results of the APEX-based proteomic profiles.
The study's authors noted that they successfully directed APEX to other cell sites, too, suggesting the same approach could prove useful for cataloging proteins in other organelles as well.
"[W]e have developed a method for mapping the proteomic composition of cellular organelles," Ting and her colleagues concluded, "using a genetically-targetable peroxidase that catalyzes the generation of short-lived, highly-reactive, and membrane-impermeant radicals in live cells."
"Our initial demonstration on the human mitochondrial matrix proteome shows that specificity is exceptionally high," they added, "because labeling is performed in living cells while membranes and other structures are still intact."