an orchestra of proteins
by Tiffany Heady

magine listening to multiple pieces of music simultaneously and trying to identify each instrument and the part that it's playing. Proteomics researchers face a similar challenge as they struggle to determine the role of each protein in the body.

Proteomics researchers work to determine what proteins are present in a cell, where they are located, how much of each protein is present, and how the proteins function. But because cells are dynamic, the protein constitution of a single cell is constantly changing, so some proteins may not even be present at certain stages of the cell cycle. If you add to this the fact that proteins are continuously modified, the task proteomics seeks to accomplish seems insurmountable.

But Christoph Borchers, assistant professor of biochemistry and biophysics and faculty director of Carolina's Proteomics Core Facility, believes that mass spectrometry (MS) is just the technology for the job. He and his colleagues have assembled a state-of-the-art automated facility, offering the latest way to look at the entire protein environment of a cell. "With this technique you can listen to an entire orchestra of proteins," Borchers says.

.: Christoph Borchers: "Music is pleasing only if each instrument plays the right notes. Our bodies work only if each protein correctly plays its specific part. With mass spectrometry, you can listen to an entire symphony." Photo by Steve Exum; click to enlarge. :.

To understand Borchers' excitement, you need to understand MS. First, a molecule is vaporized and charged, or ionized. Until 5 to 10 years ago, ionization and vaporization of fragile molecules were the Achilles heels of biological MS. Traditional techniques used heat, which could destroy peptides or proteins. But today, gentle ionization and vaporization techniques can safely transport charged peptides and proteins into the gas phase.

The mass spectrometer then measures each ion's time of flight — the time it takes for the ion to reach the detector. The time of flight is affected by both an ion's charge and its weight or mass. For instance, ions with lower weights will travel faster and will have a shorter time of flight. So the time of flight is used to determine each ion's mass-to-charge ratio, which scientists can then use to identify the peptide or protein in question.

orchers explains that MS has three distinct advantages over existing technology. MS is extremely accurate, able to distinguish a protein that weighs 1000.03 Daltons (a Dalton is the unit for protein weight) from one that weighs 1000.04 Daltons. MS is also extremely sensitive, requiring very little protein material. One of the three instruments at the Proteomics Core Facility is able to detect a femtamole of protein — a feat similar to detecting the addition of one drop of water to your backyard pool. This level of sensitivity allows researchers to use the native protein from cells instead of synthetic protein.

Finally, MS is able to provide sequence data that can be used to identify unknown proteins. Identifying all of the amino acids and their order in a given protein is referred to as protein sequencing. This process starts with digestion of the protein with an enzyme that systematically chops up the protein into smaller peptide fragments. The molecular weights of the peptides are then measured by MS so accurately that the protein can be identified by searching these masses against a protein or genome database. Like the unique grooves and coloring of jigsaw pieces, the masses of amino acid fragments indicate how the fragments can be pieced together to reveal the whole protein.

Borchers is also using MS to characterize proteins. "I call it proteomics, the second generation," he says, noting that "protein characterization is still not easy." Protein modifications such as phosphorylation and glycosylation attach additional chemical groups onto the protein, increasing its weight — but not by much. Low-weight modifications are difficult to detect by mass spectrometry. And, many of the modifications result in a negative charge on the protein, also making it difficult to detect.

"What proteomics needs to do, finally, is identify not only expressed proteins, but also identify and characterize altered proteins," Borchers says. A collaboration with the Lineberger Comprehensive Cancer Center seeks to work on some of those questions. "What we want to do is characterize the proteins of the human breast cancer cell," Borchers says. At 25,000 to 30,000 proteins, the breast cancer cell will certainly be a proving ground for proteomics.

Tiffany Heady is currently a postdoctoral student in chemistry and a freelance science writer.