Cruisin with the top down

It should probably come as little surprise that James Watson--of Watson and Crick and their vision of DNA's double-helical structure--understands proteomics' power. At a biotechnology symposium in early 2003, Watson referred to DNA as the "script" and proteins as the "actors."

Neil Kelleher, an assistant chemistry professor at the University of Illinois at Urbana-Champaign, couldn't agree more.

"The realization of our society's expectations for 21st century medicine depends on further insights into biology at the level of proteins," he says, "not just DNA." These insights will rely on "a new, dominant methodology for the collection and interpretation of proteomic data."

The Kelleher team with the Fourier-transform mass spectrometer.

Kelleher and his research team hope their "top-down" approach to identifying and characterizing proteins will become that new method. An advanced mass spectrometer, the Alliance's Condor computing system at the University of Wisconsin, and NCSA's expertise have all been integral to this nascent approach's development.

Ian Brooks, an NCSA research programmer on the project who is also a biochemist, explains the importance of Watson's distinction between actor and script: "For a simple organism--a bacteria, for example--you only need their genome [to address central questions about cell biology on a whole-system level]. For that organism, there is a one-to-one correspondence between a gene and the protein that it expresses."

"Humans are too complex," he continues. "Their gene sequence doesn't tell you what proteins are going to be made. Many proteins come from a single gene." Though there's much to be learned from the gene sequence, it tells researchers far less when they're studying mammals. In fact, the Kelleher group has found that about 10 percent of proteins are chemically different than the proteins expected when analyzing the gene sequence alone.

Schematic highlighting the differences between the Kelleher team's top-down approach to protein characterization and the more traditional bottom-up approach.
(click here to enlarge picture)

In complex critters like mammals, proteins might vary from those typically expressed due to variations in DNA transcription or RNA splicing during expression or due to chemical alterations to proteins' amino acids following production. Thus, many different proteins can result from any single snippet of genetic code. Once expressed, and in some cases altered, the proteins go about their business--whether they're enzymes that regulate biochemical activity, hormones such as insulin, or the structural basis for your body's hair, muscles, and skin.

The Kelleher team is out to characterize what are called post-translational modifications. Post-translational modifications change proteins' properties chemically by chopping up the proteins or "decorating" certain amino acids within the protein. Tacking on a phosphate, for example, fundamentally changes the protein. It might make an active enzyme of the protein or flip a molecular logic switch from 0 to 1.

According to Brooks, there are about 300 known post-translational modifications that can alter a protein and therefore influence its function or location in a cell. Formal, direct analyses have been "limited to date and totally unsystematic," says Kelleher.

A system such as the Kelleher team's would allow researchers to characterize all of the possible modifications that a specific protein might undergo. It would also allow researchers to catalog the various modification states so the next team through the breach wouldn't have to duplicate the effort.

Access Online | Posted 10-7-2003