Identifying Proteins

Today, medical clinics and research laboratories routinely identify proteins using antibodies. This requires technicians to have an antibody for each protein they wish to identify. But so far, we’ve identified antibodies for only a limited set of proteins. An alternative identification technique, recently perfected, uses mass spectrometry.

Strategies for Identifying Proteins

Proteins can be thought of as either one- or three-dimensional:

• Proteins can be identified by their one-dimensional structure — that is, their amino acid sequence. This is the strategy used by mass spectrometer-based techniques that form the bulk of today's proteomics. (There's more on this below.)
• Proteins can also be identified by their three-dimensional structures. Antibodies have a 3D shape that fits the shape of a protein (see figure on the right), somewhat like the puzzle pieces fit together in the cartoon above.

Antibodies — The Once and Future Proteomics King

In clinical practice today, proteins are always identified by antibodies. This has both good and bad aspects:

How is antibody identification good? An antibody locks on to a specific protein even when surrounded by thousands of others. So antibodies don't require proteins to be separated, as mass spectrometers do. (See the column to the right.) Antibodies are also sensitive: they can detect minute amounts of a protein. For many proteins, you can buy an antibody from a catalog.

How is antibody identification limited? Today, antibodies are not available for most proteins. Those that are available are costly ($200 - $300 per 50 micrograms). Finally, you must know precisely which protein you are looking for, so that you can get the correct antibody. But proteomics experiments frequently seek to identify unknown proteins. Today, therefore, antibodies are used in proteomics only as a confirmatory step, after mass spectrometry has identified the proteins.

Microarrays are tools that allow researchers to measure the expression of a great many genes simultaneously. Their success has led some to suggest that antibodies be arranged in protein arrays — a panel of antibodies to detect a large number of proteins simultaneously. Some protein arrays are available commercially, but they are still very limited. Most forecasts say that protein arrays covering a large percentage of proteins will be important for proteomics someday, but they are not important yet.

Mass Spectrometry Strategies for Identifying Proteins

Several mass spectrometry strategies can identify proteins:

• Tandem mass spectrometry (abbreviated MS/MS) is currently the most popular technique in research labs. Tandem mass spectrometry uses two mass spectrometers hooked together to analyze peptides — subchains of amino acids. The first machine weighs a peptide, the second identifies it.
• Protein mass fingerprinting is a somewhat older technology that uses a single mass spectrometer; it isn't as accurate at identifying proteins as MS/MS.
• Intact protein mass spectrometry looks at proteins rather than peptides. This top down approach has been successful with the high accuracy that is now possible with the (very expensive) Fourier transform mass spectrometer.

All of these mass spectrometry strategies work only on samples containing just a few different proteins. Since biological samples have hundreds or thousands of proteins, the proteins in these biological samples must be separated out into simpler samples.

Protein Separation

Cells and biological fluids such as blood have hundreds or thousands of proteins. Most protein identification methods, however, are confused when more than a few proteins are present. The mass spectrometry strategies discussed above, for example, all require separating the proteins first.

Protein scientists have spent years developing techniques for separating the proteins in a complex mixture. A good part of the lab work in proteomics consists of applying these techniques.

Electrophoresis is the separation of proteins by applying an electrical current to proteins in a gel. In the one-dimensional gel at the right, how far the proteins moved depended upon their pH. In other gels, the distance moved depends on the protein mass. In 2-D gels, proteins are separated by both mass and pH.

Protein Separation - High Performance Liquid Chromatography

High Performance Liquid Chromatography (HPLC) separates proteins as they percolate through a column packed with small particles that form a porous matrix. The proteins are dissolved in a liquid (often water) that permeates through the column. The proteins stick to the particles in the column to some degree, but they eventually wash out.

Because different proteins have different chemical properties, they wash out at different times. By exploiting these different chemical properties, proteins can be separated in several ways:

• Ion exchange, specifically strong cation exchange, separates proteins based on their pH.
• Reverse phase separates proteins based upon their hydrophobicity — how much the protein attracts or repels water.
• Size exclusion separates proteins based on their size.
• Bio-affinity separates proteins based on the degree to which proteins stick to antibodies or other substrates.

In most experiments, several of these separation techniques are combined to separate the proteins in each biological sample. For example, one common method is to:

1. Concentrate the proteins with a bio-affinity HPLC or size exclusion HPLC.
2. Separate the proteins on a 2-D gel.
3. Cut the protein into short peptides.
4. Separate the the peptides with a reverse-phase HPLC.

Another method (sometimes known as MudPIT or shotgun proteomics) is to:

1. Cut the protein into short peptides.
2. Separate the peptides with ion-exchange HPLC.
3. Separate the peptides further with reverse phase HPLC.