The Promise of Proteomics

Now that the human genome is sequenced, what's next? "The proteome," says Catherine Fenselau, professor of chemistry and biochemistry at the University of Maryland. "The proteome is a set of proteins expressed by a cell during its lifetime, and it is fast becoming a top area of interest within the biotechnology community."

Fenselau and other researchers from academia, government and industry are moving beyond genomics research into the emerging, and they say more promising, field of proteomics--the study of the expression, interaction and modification of proteins. "Genomics is important, but it is just the beginning," Fenselau says. "Now, the major challenge is to use the wealth of information that resulted from the genome sequencing project to really understand proteins and how they are related to biological function and dysfunction."

While proteomics holds great promise for diagnosis and treatment of disease, researchers in this emerging field face a huge task as they unravel the estimated 500,000 to 1 million proteins in the human body, a much larger number than the 30,000 to 40,000 genes sequenced in the Human Genome Project. Despite the challenge, Fenselau says the scientific rewards are worth the effort. "The genome is the blueprint for a building, and the proteins are the walls, floors, elevators and all of the other things that really make the building work," she says. "They are responsible for all of the life of a cell, and it is at that level where most disease states develop."

Proteomics has various applications, but Fenselau and her research team are focusing on the use of proteomics in cancer cases. They recently received a four-year grant from the National Institutes of Health to develop and apply proteomics approaches to help scientists better understand how cancer patients become resistant to certain drugs.

Using a technique called comparative proteomics, Fenselau and her colleagues are comparing the levels of proteins in drug-susceptible cancer cells to the levels of proteins in cancer cells that are resistant to drugs. Resistance is generally linked to repeat exposure to the drug, which can cause protein expression levels to change.

Biochemistry researchers at the University of Maryland are trying to understand the role of proteins in human resistance to cancer drugs. The research involves a combination of technologies, including separating proteins form cells, mass spectrometry and bioinformatrics.

In the laboratory, Fenselau and her team use a combination of technologies--from separation techniques to mass spectrometry and bioinformatics--to conduct their research. "We first grow the cells in culture dishes. Then we harvest the cells and isolate the proteins from them," says Fenselau.

To separate the individual proteins, researchers use a few different strategies. One calls for using two-dimensional gel electrophoresis, which separates proteins from each other on a gel according to the pH in one dimension and molecular weight in the other dimension. After the proteins are separated, researchers use mass spectrometers to map, characterize and identify the new proteins based on the molecular masses of the peptide fragments. Then bioinformatics software is used to compare the masses of peptides with those of known proteins.

The second separation strategy quantitatively compares drug-susceptible and drug-resistant cells. Developed in Fenselau's lab, this method involves enzymatically fragmenting the protein mixture into peptides. The mixture is then analyzed using chromatography, a process that separates the peptides based on their structure and composition. To compare the protein levels in drug-susceptible and drug-resistant cells, a heavy isotope of oxygen is introduced to the peptides from one sample. The two samples are then combined and a mass spectrometer is used to measure the ratios of normal and heavy isotopes.

"We have had promising results in two areas," says Fenselau. "We developed the new method, and we're beginning to identify the actual proteins whose levels change when the cancer cells become resistant to drugs."

Now, Fenselau and her team are planning to combine the findings from the two research areas and use the new method to identify and characterize proteins in the cancer cells. In the future, they hope to collaborate with cellular biology experts at the university to better understand the proteins that appear to play a role in drug resistance. "Understanding what they mean is the bigger picture, and that's the next step we look forward to taking," she says. --MM