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.