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
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