September 03, 2012

• Mary
  Beckman, PNNL, (509) 375-3688
• Frances
  White, PNNL, (509) 375-6904

Mass spectrometry protein assays that match sensitivity and
accuracy of antibody-based clinical tests might speed drug
discovery, basic biology research

RICHLAND, Wash. – 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.

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.

---

Reference: Tujin Shi, Thomas L. Fillmore, Xuefei Sun, Rui Zhao,
Athena A. Schepmoes, Mahmud Hossain, Fang Xie, Si Wu, Jong-Seo Kim,
Nathan Jones, Ronald J. Moora, Ljiljana Paša-Toli, Jacob
Kagan, Karin D. Rodland, Tao Liu, Keqi Tang, David G. Camp II,
Richard D. Smith, and Wei-Jun Qian, An antibody-free, targeted mass
spectrometry approach for quantification of proteins at low pg/mL
levels in human plasma/serum, Proc Natl Acad Sci U S A, Early
Edition online the week of September 3, 2012. DOI:
10.1073/pnas.1204366109 (http://
www.pnas.org/cgi/doi/10.1073/pnas.1204366109)

Tags: Fundamental
Science, Biology, Health
Science, Biomolecular
Science, Mass
Spectrometry, Proteomics

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