Activity-Based Proteomics Meets Click Chemistry

By Jason Socrates Bardi

Economic theorists have a name for what happens when an application becomes married to a single technology—they call it path dependence. Path dependence describes things like how typing is married to the standard QWERTY keyboard or how home video was long married to the VHS format.

Curiously, there is no name for the historical precursor to path dependence: where a single application lives among a frenzy of possible technology suitors. If there were such a name, it would aptly describe proteomics—an application to which many different technologies are applied, from advanced mass spectrometry to gene chips to age-old electrophoresis.

What is interesting about proteomics, the study of the expression, location, concentration and activity of specific proteins, is that it offers the possibility of looking at which proteins are specifically involved in some discrete pathology—which proteins are present in cancer cells, for instance—and many technologies are available to researchers today who want to ask such questions.

One emerging proteomics technology, called activity-based protein profiling, is being developed and applied in the laboratory of Benjamin Cravatt, who is a professor in the Department of Cell Biology and The Skaggs Institute for Chemical Biology at The Scripps Research Institute.

Activity-based profiling seeks to answer even bigger questions than more conventional proteomic approaches, such as which proteins are active in a given cancer cell.

Working at the borders of chemistry and biology, Cravatt and his colleagues have pioneered a way to survey this by developing chemical probes called "affinity labels," which have the ability to attach to the active sites of entire enzyme families in complex proteomes. These simple small chemical probes combine a reactive group, which binds to and covalently modifies the active sites of the enzymes, with a readout group—a molecular tag that can be used for the detection and isolation of the enzymes.

The idea is simple: throw these probes into living cells, let the reactive groups label the active enzymes inside, then fractionate the cells, separate the protein components, and use the readout groups to identify those that are tagged through methods like gel electrophoresis.

One of the great drawbacks of this method has been that the readout group portion of these chemical probes has been fluorescence tags and other bulky molecules that limit the probe's ability to get inside a cell and label an enzyme.

In response to this limitation, Cravatt and graduate student Anna Speers, who is a Howard Hughes Predoctoral Fellow and a Ph.D. candidate at Scripps Research's Kellogg School of Science and Technology, have extended activity-based proteomics by scaling down the size of the probes they use from a bulky fluorescent molecule to a tiny azide, which is about 10-20 times smaller than the fluorophores the Cravatt lab was using before.

Their new "tag-free" strategy for activity-based proteomics relies on using copper(I)-catalyzed azide-alkyne cycloaddition—a reaction otherwise known as click chemistry.

Click chemistry is a modular protocol for organic synthesis developed by Scripps Research Chemistry Professor and Nobel laureate K. Barry Sharpless. It relies on using energetic yet stable building blocks like azides and alkynes that will react with each other in a highly efficient and irreversible spring-loaded reaction.

Using their click chemistry proteomic probes, Speers and Cravatt can label enzyme activities in Vivo with their small azides, fractionate the cells, and then add the fluorophores, which have an alkyne arm that can readily attach to the azide labels on the tagged enzymes. Then the investigators can simply isolate the fluorescent enzymes.

In a report appearing in the latest issue of Chemistry & Biology, Speers and Cravatt showed that their technique could identify and quantify enzyme activities in living breast cancer cells. It could also discriminate between active enzymes in the living cells and inactive enzymes in the homogenate of the same cells.

The scientists also demonstrated that they could survey the activity of an inhibitor against an enzyme in Vivo. They treated mice with disulfiram, a known inhibitor of the liver enzyme aldehyde dehydrogenase-1 and demonstrated that a corresponding reduction in aldehyde dehydrogenase-1 activity could be detected.

This is significant because it demonstrates that scientists might be able to use activity-based proteomics to screen inhibitors in vivo and to determine targets in the context of living cells and organisms.

To read the article, "Profiling Enzyme Activities In Vivo Using Click Chemistry Methods" by Anna E. Speers and Benjamin F. Cravatt, see the April 2004 issue of Chemistry & Biology (p. 535) or go to http://dx.doi.org/10.1016/j.chembiol.2004.03.012.

 

Send comments to: jasonb@scripps.edu

 

 


A "tag-free" strategy for activity-based protein profiling using click chemistry.
Click to enlarge.