Mapping the living cell: New technique pinpoints protein locations, helping scientists figure out their functions

February 1, 2013 by Anne Trafton
MIT chemists have devised a way to identify which proteins are present in different compartments of the mitochondria. Credit: Jeff Martell, Hyun-Woo Rhee and Peng Zou

To get a clear picture of what's happening inside a cell, scientists need to know the locations of thousands of proteins and other molecules. MIT chemists have now developed a technique that can tag all of the proteins in a particular region of a cell, allowing them to more accurately map those proteins.

"That's a for biology—to be able to get spatially and temporally resolved molecular maps of living cells," says Alice Ting, the Ellen Swallow Richards Associate Professor of Chemistry at MIT. "We're still really far from that goal, but the overarching motivation is to get closer to that goal."

Ting's new method, developed with researchers from the Broad Institute and Harvard Medical School, combines the strengths of two existing techniques— and mass spectrometry—to tag proteins in a specific cell location and generate a comprehensive list of all the proteins in that area.

In a paper appearing in the Jan. 31 online edition of Science, Ting and colleagues used the new technique to identify nearly 500 proteins located in the mitochondrial matrix—the innermost compartment of the cellular organelle where energy is generated.

Lead authors of the Science paper are former MIT postdoc Hyun-Woo Rhee and Peng Zou, who received a PhD from MIT in 2012.

Protein labeling

Using fluorescence or , scientists can determine protein locations with high resolution, but only a handful of a cell's approximately 20,000 proteins can be imaged at once. "It's a bandwidth problem," Ting says. "You certainly couldn't image all the proteins in the at once in a single cell, because there's no way to spectrally separate that many channels of information."

With mass spectrometry, which uses ionization to detect the mass and of a compound, scientists can analyze a cell's entire complement of proteins in a single experiment. However, the process requires dissolving the to release a cell's contents, which jumbles all of the proteins together. By purifying the mixture and extracting specific organelles, it is then possible to figure out which proteins were in those organelles, but the process is messy and often unreliable.

The new MIT approach tags proteins within living cells before mass spectrometry is done, allowing spatial information to be captured before the cell is broken apart. This information is then reconstructed during analysis by noting which proteins carry the location tag.

The new system makes use of a chemical tag that includes biotin, one of the B vitamins. To label proteins with biotin, the researchers first designed a new enzyme they dubbed APEX. This enzyme is a peroxidase, meaning that it removes an electron and a proton from its substrate in a reaction known as oxidation.

Every peroxidase has different substrates, and one of APEX's substrates is biotin-phenol. When the researchers add biotin-phenol to a cell engineered to express APEX, the enzyme creates biotin-phenoxyl radicals—highly reactive molecules with unpaired electrons. These radicals quickly grab on to nearby proteins, tagging them with biotin.

To make sure the biotin is only labeling proteins in a specific part of the cell, the researchers engineer APEX so it is attached to a protein or peptide that they know is located in the area of interest. Once in the right spot, the APEX enzyme labels any other protein within a short distance of its original target.

After the proteins are labeled, the process is very routine, Ting says. Cells are dissolved, biotin-tagged proteins are extracted, and the proteins are identified by mass spectrometry.

"What you do is tag the proteins with biotin while the cell is still alive, and then you just pull out those proteins," Ting says. "Therefore you bypass all of the problems that are associated with trying to purify regions of cells and organelles, because you don't have to anymore."

The new method is an "elegant and powerful approach" to defining the protein populations of cellular organelles, says Xiaowei Zhuang, a professor of chemistry and chemical biology at Harvard University.

"If we take the analogy that an organelle is like a machine, what the method can offer us is the complete parts list of the machine, and where the parts are. Such knowledge goes a long way in helping us understand how the machine works. It is truly an important and potentially transformative technology development," says Zhuang, who was not part of the research team.

A comprehensive list

To demonstrate the technique's power, the researchers created a comprehensive list of the proteins found in the mitochondrial matrix. Most of a cell's energy generation takes place in mitochondria, as well as many biosynthetic processes.

Using the new method, the team identified nearly 500 proteins in the mitochondrial matrix. Previous attempts to map the matrix proteome by extracting that cellular compartment and then performing yielded a list of only 37 proteins. "There was no previous high-quality map of the matrix subdomain of mitochondria, and now we have one," Ting says, adding that this new wealth of information should help biologists to learn more about the functions of many of those proteins.

Already, the team has found that an enzyme called PPOX—involved in synthesizing heme, the iron-porphyrin complex found in hemoglobin—is not located where biologists had thought it was. As heme precursors move through the biosynthetic pathway, they are shuttled to different parts of the cell. Finding that PPOX is in the matrix means that there must be unknown transporter proteins bringing heme precursors into the matrix, Ting says.

"Just relocalizing things causes people to have to rethink these biosynthetic pathways and how intermediates are moved around," she says. "The molecular understanding of that pathway has to be rewritten now, based on our data."

The researchers are now investigating proteins found in another compartment of the mitochondria, the intermembrane space. They are also modifying the chemistry of the labeling system so they can use it for other tasks, such as mapping the topology of membrane proteins and detecting specific protein- interactions.

Explore further: New protein tag enhances view within living cells

Related Stories

New protein tag enhances view within living cells

February 22, 2008

The view into the inner world of living cells just got a little brighter and more colorful. A powerful new research tool, when used with other labeling technologies, allows simultaneous visualization of two or more different ...

MIT probe may help untangle cells' signaling pathways

June 27, 2008

MIT researchers have designed a new type of probe that can image thousands of interactions between proteins inside a living cell, giving them a tool to untangle the web of signaling pathways that control most of a cell's ...

Chemists design new way to fluorescently label proteins

June 1, 2010

( -- Since the 1990s, a green fluorescent protein known simply as GFP has revolutionized cell biology. Originally found in a Pacific Northwest jellyfish, GFP allows scientists to visualize proteins inside of cells ...

New technique for IDing proteins secreted by cells developed

September 25, 2012

(—Researchers from North Carolina State University have developed a new technique to identify the proteins secreted by a cell. The new approach should help researchers collect precise data on cell biology, which ...

A new glow for electron microscopy

October 22, 2012

The glowing green molecule known as green fluorescent protein (GFP) has revolutionized molecular biology. When GFP is attached to a particular protein inside a cell, scientists can easily identify and locate it using fluorescence ...

Recommended for you

Study finds 'rudimentary' empathy in macaques

December 1, 2015

(—A pair of researchers with Centre National de la Recherche Scientifique and Université Lyon, in France has conducted a study that has shown that macaques have at least some degree of empathy towards their fellow ...

Scientists overcome key CRISPR-Cas9 genome editing hurdle

December 1, 2015

Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR-Cas9 genome editing system that significantly cut down on "off-target" ...

Which came first—the sponge or the comb jelly?

December 1, 2015

Bristol study reaffirms classical view of early animal evolution. Whether sponges or comb jellies (also known as sea gooseberries) represent the oldest extant animal phylum is of crucial importance to our understanding of ...

Trap-jaw ants exhibit previously unseen jumping behavior

December 1, 2015

A species of trap-jaw ant has been found to exhibit a previously unseen jumping behavior, using its legs rather than its powerful jaws. The discovery makes this species, Odontomachus rixosus, the only species of ant that ...


Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.