PNNL researchers developed a new technique using mass spectrometers, shown here, that matches the sensitivity and accuracy of antibody-based clinical tests to identify protein biomarkers associated with cancer and other diseases. The new technique, called PRISM, could speed drug discovery and basic biology research. Image: EMSL |
Combining
two well-established analytic techniques and adding a twist identifies
proteins from blood with as much accuracy and sensitivity as the
antibody-based tests used clinically, researchers report this week in
Proceedings of the National Academy of Sciences Early Edition online.
The technique should be able to speed up development of diagnostic tests
and treatments based on proteins specific to certain diseases.
The
team of scientists at the Department of Energy’s Pacific Northwest
National Laboratory found that their technique, called PRISM, performed
as accurately as standard clinical tests known as ELISAs in a
head-to-head comparison using blood samples from cancer patients. The
tests measure biomarkers, proteins whose presence identifies a disease
or condition.
“Clinical
tests have almost always used antibodies to measure biomarkers, because
antibodies can provide good sensitivity,” said PNNL bioanalytical
chemist Wei-Jun Qian, lead author on the study. “But it often takes a
year and a half to develop antibodies as tools. Antibody development is
one of the bottlenecks for new biomarker studies in disease and systems
biology research.”
Qian,
Tujin Shi, Tom Fillmore and their PNNL colleagues worked out the highly
sensitive PRISM using resources at DOE’s EMSL, the Environmental
Molecular Sciences Laboratory on PNNL’s campus. The result is a simple
and elegant integration of existing technologies that solves a
long-standing problem.
The competition
Researchers
have long wanted to use mass spectrometry to identify proteins of
interest within biological samples. Proteins are easy to detect with
mass spec, but it lacks the sensitivity to detect rare proteins that
exist in very low concentrations. Scientists use antibodies to detect
those rare proteins, which work like a magnet pulling a nail out of a
haystack.
Antibodies
are immune system molecules that recognize proteins from foreign
invaders and grab onto them, which allows researchers to pull their
proteins of interest out of a larger volume, concentrating the proteins
in the process. Because antibodies recognize only one or a couple of
proteins, researchers have made treatments and tools out of them. Drugs
whose generic names end in “-mab” are antibodies, for example.
For
research purposes, the modern laboratory can produce antibodies for
almost any protein. But that development process is expensive and
time-consuming. If you have a new biomarker to explore, it can take
longer than a year just to create an antibody tool to do so.
To
get around the need for an antibody, Qian and the team concentrated the
proteins in their samples another way. They used a common technique
called high performance liquid chromatography, usually shortened to
HPLC, to make the proteins about 100 times as concentrated as their
initial sample. While an excellent step, they also had to find their
protein of interest in their concentrated samples.
So they sent in a spy, a protein they could detect and whose presence would tell them if they found what they were looking for.
With
a potential biomarker in mind, the team made a version that was
atomically “heavier.” They synthesized the protein using carbon and
nitrogen atoms that contain extra neutrons. The unusual atoms added
weight but didn’t change any other characteristics. The heavier versions
are twins of the lighter proteins found within the blood, cells, or
samples. Although the twins behave similarly in the analytical
instruments, the heavier twin is easily found among the sample’s many
proteins.
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After
adding the heavy version to the samples, the team sent the sample
through the instrument to concentrate the proteins. The instrument spit
out the sample, one concentrated fraction at a time. The fraction that
contained the heavy biomarker was also the fraction that contained its
twin, the lighter, natural protein. From this fraction, the team could
quantify the protein.
Protein spectrum
To
prove they could use PRISM this way to find very rare proteins, the
team spiked blood samples from women with a biomarker called prostate
specific antigen, or PSA, that only men make. The team found they could
measure PSA at concentrations about 50 picograms per milliliter. While
typical of the sensitivity of ELISA tests, it represents about 100 times
the sensitivity of conventional mass spectrometry methods.
“This is a breakthrough in sensitivity without using antibodies,” said Qian.
Then
they tested PSA in samples from male cancer patients and found PRISM
performed as well as ELISA. Interestingly, PRISM measured three times
the amount of PSA than the ELISA assay did. This result suggests that
antibody-based ELISA tests fail to measure all of the forms of the
biomarker. This is likely due to the fact that antibodies don’t
recognize all the different forms that proteins can take, Qian said,
whereas PRISM measures the total amount of protein.
In
addition to its sensitivity, PRISM requires only a very small sample of
blood or serum from the patient. The team used only 2 microliters of
the cancer patients’ sample, a volume that would easily fit inside this
small printed “o”.
One
drawback to the technique, however, is how many biological samples can
be tested at once. Researchers want to test thousands, and
antibody-based methods allow such high-throughput testing. But PRISM can
only test several hundred samples per study. However, with the time
researchers save not developing antibodies, the technique might still
put them ahead in biomarker development.
For
basic biology research, Qian said the method will be useful for
studying biological pathways in cases where scientists need to
accurately quantify multiple different proteins.
This
work was supported by the National Institutes of Health New Innovator
Award and a Department of Energy Early Career Research Award to Wei-Jun
Qian.
Source: Pacific Northwest National Laboratory
Filed Under: Drug Discovery
