September 08, 2000
Protein Chips Offer Powerful Method for Probing Protein Function
Using microscope slides, precision robots and other off-the-shelf
equipment, researchers have created protein microarrays that can
measure the function of thousands of proteins simultaneously. These
"protein chips"–which are counterparts to the much publicized
"gene chips" that reveal the activity of thousands of genes–will
propel the next wave of proteomics research.
According to the researchers, the technique will enable rapid
screening of thousands of small-molecule drug candidates to determine
their potential to affect specific proteins. And ultimately, the
technique will allow scientists to create protein "snapshots" of
cells–profiling the massive number of enzymes and other proteins
in their various forms.
“Profiling proteins will be invaluable, for example, in distinguishing the proteins of normal cells from early-stage cancer cells, and from malignant, metastatic cancer cells that are the real killers.”
Stuart L. Schreiber
In an article published in the September 8, 2000, issue of the
journal Science, Howard Hughes Medical Institute investigator
Stuart L. Schreiber and Gavin MacBeath, both at Harvard University,
reported that they had successfully developed and tested protein
microarrays. Each microarray contained more than 10,000 spots of
protein that were robotically deposited on the surface of a common
glass microscope slide. The technique preserved the function of the
delicate proteins, which the researchers demonstrated by showing that
the deposited proteins reacted with other proteins and small
molecules.
"We took our cue from the DNA microarrays that are being used so
successfully to measure gene activity on a genome-wide basis, in the
form of messenger RNA levels," said Schreiber. "But then the question
arises how much are we missing by only looking at RNA levels? And
clearly the answer is that there's a great deal going on in terms of
the proteins in cells."
To develop a microarray method for measuring proteins that could be
used easily in other laboratories, MacBeath and Schreiber employed
equipment and materials readily affordable by academic laboratories.
"We are particularly proud that we were able to develop a technique
that can be carried out in a typical university environment under
conditions compatible with a typical university research budget," said
Schreiber.
In creating the protein chips, the scientists used a
contact-printing robot developed earlier by HHMI investigator Patrick O. Brown at
Stanford University. The robot precisely delivers tiny droplets of
liquid protein?each the width of a human hair?to microscope slides. The
robot placed liquid protein samples on microscope slides at a density
of 1,600 spots per square centimeter. The protein samples were made to
adhere to the glass slides by coating the slides with an
aldehyde-containing reagent that attaches to primary amines, chemicals
that are commonly found in proteins. The scientists also took measures
to prevent evaporation and denaturation of the proteins, thereby
ensuring that the proteins on the slide would retain their natural
shape and activity.
The scientists performed three kinds of experiments to demonstrate
that their protein microarrays could be used to determine the
functionality of proteins. In one set of experiments, the researchers
showed that the arrays could detect protein-protein interactions. They
created microarrays of proteins and treated those microarrays with
fluorescently labeled proteins that were known to attach to the
proteins on the slide. The fluorescent spots that were clearly visible
on the slides indicated the proteins had attached to one another.
In another set of experiments, the scientists showed that the
microarrays could reveal interactions between enzymes and their
substrates, molecules upon which the enzymes act. The researchers
treated an array of kinases with radiolabeled kinase substrates. When
the treated microarrays were "developed" in a photographic emulsion,
the radiolabels were detectable as spots on the microarrays.
In a third type of experiment, the scientists demonstrated that the
protein microarrays could be used to detect small molecule-protein
interactions by incubating the protein microarrays with small molecules
in solution. Earlier, the scientists had created arrays of small
molecules (small molecule microarrays) using a technique called
diversity-oriented organic synthesis. When the arrays were treated with
fluorescently labeled proteins that contained target receptors that
interacted with the molecules, the microarray spots revealed that there
was normal binding.
"We believe that both protein and small molecule microarrays can be
used for two fundamentally different purposes," said Schreiber. "And
these initial experiments demonstrate the simplest one–analyzing
the functionality of proteins such as binding.
"This is only a starting point," he emphasized. "The most important
future application of this technique will be in profiling the proteins
in cells under different conditions, just as RNA profiling reveals the
relative levels of RNA present in a cell.
"Profiling proteins will be invaluable, for example, in
distinguishing the proteins of normal cells from early-stage cancer
cells, and from malignant, metastatic cancer cells that are the real
killers." However, noted Schreiber, protein profiling will prove far
more difficult than RNA profiling.
"The proteome–that is, the cell's array of proteins–is
more complex than the genome," he said. "Although one gene may encode
one protein, those proteins are modified in many ways after they are
constructed. So, each gene product may result in dozens of proteins
that have been rearranged, fragmented or chemically modified to produce
a slightly different activity. And there is every reason to believe
that these modified proteins are going to be key elements to
understanding function and eventually physiology.
"We are optimistic that in a short time we will meet the technical
challenges that will enable protein profiling with this technique,"
said Schreiber.
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