Indiana University Research & Creative Activity

The Art and Science of Medicine

Volume XXVI Number 1
Fall 2003

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3D image of protein structure
a protein structure
courtesy U.S. Department of Energy Human Genome Program

Mu Wang, standing, and Frank Witzmann
Mu Wang, standing, and Frank Witzmann
Photo 2003 Tyagan Miller

mass spectrometer
Mass spectrometer
courtesy Proteomics Core Facility, IU School of Medicine

Honing the Proteome

by Eric Schoch

As many as 150,000 Americans, and countless thousands more people around the world, struggle with neurofibromatosis, which causes tumors to grow on nerve tissue. It's the most common disease caused by the mutation of a single gene and can leave people disfigured, in pain, blind, deaf, or struggling with malignancies.

Except for surgery to remove threatening tumors, there is no treatment, there is no cure.

Wade Clapp, associate professor of pediatrics and of microbiology and immunology, and his colleagues at the Indiana University School of Medicine are working to change that. One of their areas of focus is the mast cell, an immune system component that appears to stimulate the growth of the tumors by secreting protein messengers.

The question is, what are those proteins? To answer, Clapp has turned to one of the hot new technologies at IU, one that has emerged from the hoopla of the Human Genome Project--proteomics.

And Clapp isn't alone. Just weeks after the School of Medicine's new Biotechnology Research and Training Center opened, the backlog of "orders" for its proteomics core facility labs had mushroomed, and turnaround time had grown from two weeks to more than a month.

Part of this was due to the availability of money--investigators have been putting funding for proteomics work into their budgets the past couple of years, says Mu Wang, director of the proteomics core facility, and assistant scientist and assistant professor of biochemistry and molecular biology. But it also represents the realization that while the sequencing of the human genome got the publicity, the answers to many of today's biomedical research questions are to be found in proteins.

That's why the proteomics core was one of nine areas funded by a $105 million grant from the Lilly Endowment that created the Indiana Genomics Initiative in 2000. The core builds on expertise and collaboration at the School of Medicine in Indianapolis and on the IU Bloomington campus, where experts in analytical chemistry are laying the groundwork for the next generation of proteomics research.

Wang emphasizes that proteomics is a tool, not a scientific field. Biology is a field, biochemistry is a field. Proteomics is a tool you use to do research in those fields, studying proteins. But as tools go, it's a good one.

"Before, people were looking at individual proteins, looking at the interactions of two proteins. Now you are looking dozens of proteins at the same time, and you can identify them," says Wang.

"Eventually you need to validate the things you discover from using this tool," he continues. "This tool cannot do the clinical trial for you. It can help you do the clinical trial, it can help you answer important biological questions. It won't be able to say if this protein is the one really doing anything.

"This is technology applied to solve a biological problem."

The problem is a complicated one, because the proteome--the universe of proteins in a cell at any given moment--is a complicated universe.

Proteins are the molecules that do the heavy lifting in our cells. They're the building blocks, the messengers, the contractors, the bosses, and the worker bees of all the activity. Proteins are made when genes give the orders. It's the central dogma of biology: A sequence of DNA-a gene-is transcribed into RNA. The RNA moves out of the nucleus, and the RNA sequence is translated into a protein.

DNA to RNA to protein. Simple? Only as dogma.

First, while genes are composed of four bases, proteins are made up of 20 amino acids. Second, except for the occasional mutation, the genome--the full set of genes--is pretty much static. A person's genome changes very little over time. But the set of proteins inside any one cell--the proteome--is constantly changing.

The proteome of a muscle cell is different than the proteome of a brain cell. The proteome of a muscle cell now is different than the proteome of a muscle cell later. And the proteome of a healthy cell is different than the proteome of a diseased cell. Protein production, usage, and disposal all change as conditions change.

And to add another level of complexity: when proteins come off the production line, their chemical makeup isn't fixed. Other molecules, such as phosphates, may be attached. These and other "post-translational modifications" can change a protein's shape, how it functions, or whether it's used at all.

The goal is to figure out how all this works, to learn which proteins come and go when, say, a cell becomes cancerous, and why, or to look for markers, or for drug targets. Everybody wants to use the proteomics tool now, says Wang. "Hopefully in a couple of years, five years, we will see important discoveries coming out of this."

Wang focuses his personal research on the systems that cells use to repair DNA. Mangled DNA, whether caused by smoking, cosmic rays, or slipups in mitosis, can lead to cancers or other problems that cells try to head off with proteins specifically designed to fix DNA. There's a downside to this: cancer cells can become expert at fixing the DNA damage caused by chemotherapy drugs. Wang wants to find out how to block those repair mechanisms so that chemotherapy drugs will be more effective.

Wang's expertise is in the use of mass spectrometers, expensive machines that use the principles of physics and chemistry to help scientists identify proteins after breaking them into pieces.

But the first step is often a seemingly lower tech one--two dimensional gel electrophoresis. It's a process mastered by Frank Witzmann, scientific adviser to the proteomics core and professor of cellular and integrated physiology and of biochemistry and molecular biology at the Medical School. According to David Clemmer, chairman of IUB's chemistry department and a proteomics expert, Witzmann has turned the 2D gel into an art form.

Witzmann says he merely learned well from the masters, Norman and Leigh Anderson, the father-son team who developed the process while working at Argonne National Laboratory in Illinois. Witzmann's specialty takes advantage of two properties of proteins: they vary in size, or molecular weight, and they vary in charge (isoelectric point or pI).

Those properties make 2D gels essentially a two-step process. First, a solution containing sample proteins is placed in a thin strip of gel. When an electrical charge is applied to the strip--positive on one end, negative on the other--the proteins migrate to the spots on the strip where their positive and negative charges are balanced. This has the effect of separating the proteins according to their pI--acidic proteins to the left, basic proteins to the right.

In the second step, a tube is placed at the end of a slab of gel, the proteins are treated to give them all a negative charge, and power is applied again. The proteins move through the gel toward the positive electrical pole, but the smaller proteins can move through the gel faster. In the end, you have a rectangle covered with dots (which can be seen because they've been treated with a blue dye).

The resulting pattern is one that an experienced researcher can read, much like an experienced skywatcher can pick out stars and constellations at night. But an experienced astronomer, with the right equipment, can do more--he or she can analyze the same section of sky over time and spot the changes that indicate a moving planet or an exploding star.

That's the kind of thing that Witzmann and his colleagues look for. For example, dots (or proteins) in a sample of diseased tissue that weren't there in the sample of healthy tissue. Or the change (the post-translational modification) that occurs after a drug is given.

"If you conduct the 2D gel runs the way we do them, taking great care that every gel is run the same way as the next one, doing 20 at a time, then you can do valid comparisons between the gels you run this week and the ones you run next week, or a month from now," says Witzmann.

"And we happen to run pretty darn good gels."

He and his team have run as many as 224 gels on 224 samples in a single experiment, with each gel revealing 1,500 to 2,000 proteins, a process that produces a huge amount of data.

Still, there are times when the proteins of interest are present in amounts too small to be detected by standard techniques. So Witzmann is working on methods such as subcellular fractionation--looking at proteomes of particular segments of a cell. Think of a bathtub after a child's bath. Initially, the tops of only a few toys appear in the water. Drain some of the water, though, and more toys will poke through the surface of the water.

The patterns of 2D gels can reveal much, but often there's a next step. To identify a particular protein, the dot in the gel can be snipped out, treated, and analyzed in the premier tool of proteomics, the mass spectrometer.

Development of new mass spectrometry techniques has revolutionized scientists' ability to study proteins--half of the 2002 Nobel prize in chemistry went jointly to two scientists for their development of new mass spectrometry methods (the other half went to a scientist for his development of nuclear magnetic resonance spectroscopy).

Put simply, a mass spectrometer is an expensive, high-tech device used to weigh very tiny things, like proteins. It does this by turning compounds into charged particles (ions) and then subjecting them to electric or magnetic fields. How an ion reacts to the field will be determined by its mass, and that lets it be measured. The first mass spectrometer was developed in the first decade of the 20th century, making one of the key technologies for this century's biomedical research revolution a fancy scale developed more than 100 years ago. Of course, the technology has improved a bit since then.

What, exactly, does a mass spectrometer do?

In Bloomington, David Clemmer, professor of chemistry and Robert and Marjorie Mann Chair of the department, starts his "Mass Spec 101" talk by imagining he's throwing a series of balls across his conference room, supposing he's mastered the ability to throw each one exactly the same way, with the same amount of force. So if he throws a heavy stone ball and a lighter foam ball, the foam ball will go farther (we'll forget about the effects of air resistance here). Balls tossed in the Clemmer Throwing System would line up according to their mass, heaviest balls closest and lightest balls further away. In other words, the mass of a ball could be calculated based on how far it traveled.

So far, so good. But think of two balls with the same mass, one made of stone, for example, and the other made of glass. Two very different balls, but the Clemmer Throwing System can't tell them apart by their mass alone. So consider a second step: Take a bat, and smack the balls. The stone ball breaks into a few chunks. The glass ball shatters into a zillion pieces. Now we have more information, and we can tell by the breakage patterns which ball was rock and which one was glass.

Which brings us back to proteins.

The clump of proteins taken from the gel can be analyzed in the mass spectrometer by ionizing them-- attaching a proton to the proteins to give them an electrical charge. By shooting the ionized proteins through a gas, they can be forced to break apart. The proton attached to each protein will stick with one of the broken pieces. Those "daughter ions" are subjected to the magnetic field and can be measured. By analyzing the patterns created by the daughter ions, researchers can start piecing together the sequence of amino acids the make up the original protein.

For Clemmer and his colleagues in Bloomington, the challenge is to develop ever more precise methods of mass spec analysis. And despite advances in technology, in software, and in researchers' analytical abilities, we're just starting out on this scientific trip, says Clemmer.

"We're like Galileo was when he was looking up at the stars with the first telescope," Clemmer says. "We're seeing the first round of the proteome. And the first round is all the things we couldn't see with our naked eye. Now we can see the moons of Jupiter."

Eric Schoch is a science writer at the IU School of Medicine.

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