The Case for Activity-Based Proteomics
By Jason Socrates
Bardi
No doubt owing to the success and hype of the Human Genome
Project, several other "'omes"—the glycome, the transcriptome,
the proteome—have been popping up in the English lexicon,
like so many decorative statues on a green lawn.
These other 'omes are no mere ornaments, though. As scientific
endeavors go, unraveling the secrets of the carbohydrate in
the human glycome, the proteins in the human proteome, and
the mRNA in the human transcriptome follow naturally from
the human genome. In fact, understanding them may even be
necessary for the realization of the promise of the human
genome—that mapping will reveal much about human biology,
including possible targets and tools for addressing human
disease.
Perhaps the most immediately important of these post-genome
'omes is the proteome, which by some estimates is at least
an order of magnitude more vast than the human genome. With
multiple, alternative RNA splices, post-translational modifications,
and protein folding controlled by localization, protein interactions,
cofactors, temperature, pH, and a dozen other factors, the
40,000 or so genes in the human genome multiply into millions
of possible protein forms and states in the cell.
What is really interesting is to look at a portion of the
proteome—those gene products that are specifically involved
in some discrete pathology. What could offer more insight
than looking at the protein "profile" of a cancerous cell
compared to a normal cell, of a cancerous cell soaked in a
drug compared to one that is not?
These are applications within the scope of proteomics, which
attempts to elucidate the actual expression and action of
the genome in different cell types and states. One important
aspect of this is where and when the proteins are working
in the human body, and how they are turned on or shut off
in certain cell types, times, or conditions like infection
or cancer.
A new proteomics technology, which is being developed and
applied by two investigators at The Scripps Research Institute
(TSRI) seeks to answer an even more profound question—which
proteins are active in a given cell, tissue, or disease
state?
"It's a chemical approach to understanding protein function,"
says Cell Biology Associate Professor Benjamin Cravatt, who
began developing the approach about three years ago. Soon
afterwards, he began collaborating with his colleague and
fellow investigators in the Skaggs Institute for Chemical
Biology, Chemistry Associate Professor Erik Sorensen.
The Problem With Proteomics
Generally, proteomics is still an application in search
of its technology. While there are powerful methods for the
global analysis of mRNA levels in cells based on gene chip
technology. There are also tools for looking at proteins—using
two-dimensional electrophoresis coupled with mass spectrometry,
for instance. These techniques have lacked luster when it
comes to profiling protein activities, though. Methods that
are based on profiling expression levels may not be sensitive
to changes in the activity of a protein. And methods that
do directly look at protein activity do not necessarily do
so in the native state of the cell.
Current proteomics methods may also miss proteins that are
only expressed in low abundance, which may be exactly the
proteins that are of interest. Most of the proteins expressed
in a cell, after all, are structural and housekeeping proteins.
And they may miss proteins that are associated with membranes,
proteins with extreme isoelectric points—either very
basic or very acidic proteins—and proteins that have
very high or very low molecular weights.
Cravatt believes that activity-based profiling of proteins
expressed in living cells is the next step
"This is a way to accelerate the process [of finding] active
enzymes that correlate with disease [and] circumvent major
challenges that face proteomics technologies," says Cravatt.
Based on Fundamental Enzymology
The basis of Cravatt's technique is thoroughly grounded
in over a half-century of classical enzymology, the biochemical
field concerned with the activity and rates of catalytic biological
molecules.
Over the decades, biochemists have advanced their understanding
of various enzymes by developing chemical probes called "affinity
labels." These are simple, small chemicals with a reactive
moiety that has the ability to attach to the active sites
of protein enzymes. These chemical moieties are used to try
to understand the mechanism through which enzymes work.
Active-site proteomics takes this classical approach a step
further. Rather than employing chemical probes to profile
one enzyme, the technology uses probes to assay the activity
of entire enzyme families in complex proteomes. These probes
combine a reactive group that binds and covalently modifies
the active sites of many members of a given enzyme class,
with a chemical (e.g., fluorescent) tag for the detection
and isolation of reactive enzymes.
The idea is simple: throw these probes in together with
different types of cells, let them label their enzyme targets,
and then do a separation. Usually this separation involves
first splitting cells into the extracellular fraction (which
contains the secreted proteins), the lipidic fraction (containing
the membrane-bound proteins) and the cytosolic fraction (with
the soluble, intracellular proteins). Then the tagged enzymes
in each portion can be further separated and identified by
time-tested methods like running a gel.
By taking these labels and applying them in cases where
there is some phenotype of interest—a metastatic cell,
for instance—activity-based proteomics can give a readout
of which proteins in the cell are active and which are not.
Simple.
But nothing is ever that simple.
The real trick is to find the right probes. The best experimental
design is to cover the maximum number of enzymes in a cell
with the minimum number of what they call "promiscuous" probes.
The point is not necessarily to look at the absolute activity
of proteins in the mix but to interrogate broadly and see
whether proteins are on or off.
Luckily, when Cravatt started developing the technology,
he already had one such probe—an affinity agent for a
family of enzymes called the serine hydrolases. He had been
using the probe in the course of his studies on the fatty
acid amide hydrolase (FAAH) and other members of the serine
hydrolase family. It was known that the FAAH probes were promiscuous
and reacted broadly with other serine hydrolases, and it was
a relatively simple matter to doctor these known affinity
labels with readout tags.
"The beauty of it is that it reacts broadly within that
family and it tends not to react with enzymes of other classes,"
says Sorensen.
So far, they have begun using the technology with this probe
to study melanoma, breast carcinoma, and ovarian carcinoma
cells. They are even able to detect proteins at the femtomole
level—as few as a hundred or so copies of per cell.
Activity-Based Profiling of Cancer Lines
By far the most interesting application of the technology
is comparative profiling, which aims to detect disease-related
protein activities and to establish the identity of proteins
that are involved in the pathogenesis of diseases by comparing,
for instance, the active proteins in tumor cells to normal
cells.
In what Cravatt calls the first practical application of
the new technology, a paper in this week's Proceedings
of the National Academy of the Sciences describes the
application of the approach to cancerous cells.
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