ScienceDaily (Sep. 13, 2000) — September 8, 2000 — 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.
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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