Mining for proteins

Techniques for isolating and identifying proteins

Bill Snyder
Published: February, 2003

Photo by Anne Rayner
Proteomics is a technology-driven field. The photo at left, for example, shows proteins separated by high-performance liquid chromatography being sprayed into a tandem mass spectrometer, which will further separate them according to their “mass-tocharge” ratios. Here is a sampler of other important technologies:

Two-dimensional gel electrophoresis 

This technique separates proteins across a gel, first according to their charge (isoelectric point) and then by their size (molecular weight). After separation the gels are stained, revealing dark spots where the proteins have concentrated. Spots of interest can be extracted from the gel and analyzed to identify the proteins.

In recent years several advances have improved the accuracy and sensitivity of 2-D gels. More reliable analytical methods, including robotic machines, have replaced hand-processed gels. By tagging proteins from two separate tissue samples with different fluorescent markers, researchers now can compare them on the same gel.

“This allows us to quantify the increase or decrease of a given protein,” in different stages of cancer, for example, says David Friedman, Ph.D., director of the proteomics laboratory in the Vanderbilt Mass Spectrometry Research Center. “It is very powerful at imaging thousands of proteins at a given time, yet it is biased toward more abundant proteins in the cell.”

Mass spectrometry

Mass spectrometry is becoming an increasingly valuable and versatile technique for identifying proteins, studying interactions between proteins, and mapping the location of proteins within tissue.

Ordinarily, proteins must be “cleaved,” or broken into manageable pieces called peptides, before they can be “weighed” in the mass spectrometer. The peptides are “ionized,” or given an electrical charge, so they can be propelled by an electric or magnetic field through a vacuum toward the instrument’s detector.

From the motion of the peptides, the spectrometer calculates their “mass-to-charge” ratio, which is related to their molecular weight, and generates a spectrum of the relative amounts (or intensity) of all the peptides that make up the original protein. The resulting peak-and-valley pattern of the mass spectrum is the protein’s unique “fingerprint.”

Techniques developed in the 1980s have greatly improved the ability of mass spectrometry to analyze large, intact proteins and complex protein mixtures.

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