Aug 7 2020
A new technique developed by scientists from the University of Wisconsin–Madison (UW–Madison) integrates high-precision protein measurement with sticky nanoparticles to capture and study a common marker associated with heart disease, revealing details that were not accessible before.
Ying Ge. Image Credit: University of Wisconsin–Madison.
Called nanoproteomics, the new technique effectively records and quantifies numerous forms of the protein, known as cardiac troponin I, or cTnI for short. This biomarker of heart damage is being used to help diagnose various kinds of heart diseases, including heart attacks.
An effective test that detects the changes in the cTnI protein may someday provide physicians with a better potential to detect heart disease, which is known to be the leading cause of death in the United States.
The study was headed by Ying Ge, Professor of Cell and Regenerative Biology and Chemistry from UW–Madison; Song Jin, Professor of Chemistry; and Timothy Tiambeng and David Roberts, chemistry graduate students. The study was published in the Nature Communications journal on August 6th, 2020.
The team has now planned to apply the novel technique to correlate the numerous forms of the cTnI protein with certain heart diseases to advance the development of innovative diagnostic tests.
An antibody-based test, known as ELISA, is being used by physicians to help detect heart attacks based on increased concatenations of the cTnI protein in the blood sample of the patient.
Although the ELISA test is sensitive, individuals can have elevated levels of the cTnI protein in their blood even when they do not have heart disease. This can lead to unwanted and costly treatments for patients.
So we want to use our nanoproteomics system to look into more details at various modified forms of this protein rather than just measuring its concentration. That will help reveal molecular fingerprints of cTnI from each patient for precision medicine.
Ying Ge, Professor, Department of Cell and Regenerative Biology and Chemistry, University of Wisconsin–Madison
Professor Ge is also the director of the Human Proteomics Program at the UW School of Medicine and Public Health.
Quantifying low levels of proteins—such as cTnI—in the blood represents a traditional needle-in-a-haystack issue. Meaningful and rare biomarkers of a disease are fully overwhelmed by diagnostically useless and common proteins found in the blood.
Antibodies are used by present-day techniques to both enrich and trap proteins in a complex sample to detect and measure them. But antibodies have batch-to-batch differences, are costly, and can lead to unreliable results.
Therefore, to capture the cTnI protein and resolve some of the restrictions presented by antibodies, the team developed magnetite nanoparticles, that is, a magnetic form of iron oxide. They connected these nanoparticles to a peptide of 13 amino acids that were developed much earlier to specifically attach to the cTnI protein.
When the peptide binds to the cTnI protein in a blood sample, the nanoparticles can be gathered collectively with the help of a magnet. Peptides and nanoparticles are easily produced in the laboratory, rendering them consistent and economical.
By applying the nanoparticles, the research team successfully enriched the cTnI protein in tissue and blood samples of humans. They subsequently employed sophisticated mass spectrometry, which is capable of distinguishing different forms of proteins by their mass, to achieve a precise measurement of the cTnI protein and also to evaluate the numerous altered forms of the protein.
The cTnI protein, similar to several proteins, can be altered by the body based on factors like environmental changes or an underlying disorder. With regard to the cTnI protein, the body adds numerous numbers of phosphate groups—tiny molecular tags that could alter the role of the cTnI protein. Such differences are mild and difficult to monitor.
But with high-resolution mass spectrometry, we can now ‘see’ these molecular details of proteins, like the hidden iceberg beneath the surface.
Ying Ge, Professor, Department of Cell and Regenerative Biology and Chemistry, University of Wisconsin–Madison
Tiambeng and Roberts both decided to test whether the numerous forms of the cTnI protein present in the patient blood samples could be possibly distinguished.
Hence, they spiked the blood serum using proteins obtained from donor hearts that were diseased and normal, or obtained from a dead donor. The duo subsequently applied the nanoparticles to capture the cTnI protein and quantified the protein through mass spectrometry.
As expected, the researchers could clearly visualize varying patterns in the cTnI types prevailing in each type of heart tissue. They observed that the healthy hearts contained plenty of the cTnI protein with numerous groups of phosphate attached, for instance, while the diseased hearts had cTnI in which there were fewer phosphate groups. In the post-mortem heart, the cTnI protein was broken into fragments.
Although the study is still a proof-of-concept and additional studies will be required, the researchers believe that this potential to correlate a pattern of cTnI changes with heart health may one day lead to a novel diagnostic tool to help patients who visit the hospital with suspected heart disease.
The team has now filed a patent application on the novel technology via Wisconsin Alumni Research Foundation.
We like to think a future blood test based on our work here could be complementary to the current ELISA test. In the future, when ELISA shows an elevated cTnI level, your doctor might order a comprehensive nanoproteomics test to determine whether it is caused by heart disease or not, and identify different types of heart disease, for more precise treatment while avoiding unnecessary care and expense for patients.
Song Jin, Professor of Chemistry, University of Wisconsin–Madison
The study was funded by the National Institutes of Health (grants R01 GM117058, R01 GM125085, R01 HL096971, S10 OD018475, T32GM008505, and T32 HL007936-19).
Journal Reference:
Tiambeng, T. N., et al. (2020) Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum. Nature Communications. doi.org/10.1038/s41467-020-17643-1.