Milestones in Mass Spectrometry

When an architect presents house blueprints to a construction crew, the house is far from being considered complete and habitable. Construction workers build the house according to the set of blueprints, and then the plumbers and interior decorators finish the job.

Much like the building of a house is helped along by blueprints, DNA maps out human development. UW researchers are using mass spectrometry techniques to help decipher those house plans for the human body.

	DNA encodes some 30,000 human proteins.

"DNA is like a set of blueprints," explained Dr. Ken Walsh. "It's one thing to have the blueprints and another thing to have a finished house. Blueprints describe how to build a house, but the house, a human cell in this case, is built largely of proteins selected from a DNA-encoded list of 30,000. Most of these proteins need to be chemically trimmed before they can serve the cell."

Walsh, professor emeritus of biochemistry, joined the UW faculty in 1959 after receiving his Ph.D. in biochemistry from the University of Toronto. He was chair of the Department of Biochemistry from 1992 to 2000, when he retired In September 2002 the International Association for Protein Structure Analysis and Proteomics presented the Pehr Edman Award to Walsh for his contributions to protein chemistry, protein structure analysis, and proteomics.

"Proteins are the working machines of the cell," said Walsh. "Heart cells display different proteins from brain cells, and so the cells differ in function. The challenge is to identify which proteins actually occur inside each kind of cell. With 30,000 possibilities, fast methods are necessary."

By 1980 DNA sequencing was replacing the Edman degradation, the classic, although laborious, way to determine amino acid sequences of proteins by taking them apart one amino acid at a time.

"I did Edman degradations for 30 years," said Walsh. "When DNA sequencing came on the scene, we at the UW began to focus on other ways to characterize proteins, and the mass spectrometer became the tool of choice."

		A mass spectrometer is a device researchers employ to determine the mass of an individual molecule in daltons, the mass of one hydrogen atom. Protein molecules typically contain 200 to 2,000 amino acids and have masses ranging from 20,000 to 200,000 daltons. Mass spectrometry is ideal for two critical tasks, Walsh said: identifying a protein, and probing its functions by examining its post-translational modifications. A ribosome, for instance, consists of many different proteins that are bound together, all of which can be identified by their mass using mass spectrometry. Researchers ask how these proteins become chemically modified and how they act together to accomplish their physiological goal.
A protein model.

"It's one thing to translate the genetic information," said Walsh "It's quite another to assemble mature, functioning proteins. Back to the house analogy - a construction company can build a house from blueprints, but the house is not a functioning residence until the company put locks on the doors and toilets on the floors. We focused on the chemical changes in the proteins that occur after they are translated from the DNA, the so-called post-translational modifications."

The mass spectrometer became the ideal analytical tool, Walsh said, because all post-translational modifications either add mass, like a phosphate group, or they take away mass, like a segment of the protein. For instance, proinsulin only becomes insulin when an enzyme breaks some molecular bonds and removes a segment, thereby decreasing its mass. From a change in the mass of a protein, or a fragment of it, the nature of the attached group or removed segment can be deduced, and much can be learned of the function or control of the protein.

"With a mass spectrometer you can actually see the change in molecular mass," said Walsh. "For instance, Krebs and Fischer's Nobel-Prize winning discovery of phosphorylation in the 1950s would have been easier by mass spectrometry. When a phosphate group attaches to a protein an 80-dalton increase in the mass of a segment of a protein signals that it has been phosphorylated."

Walsh's lab at UW closed in 2000. His mass spectrometers are still available in the School of Pharmacy and the Department of Chemistry.