Researchers have discovered a blood signature of protein isoforms that could potentially predict which patients may reject a new organ transplant, helping inform therapeutic decisions. The findings of this study are reported online in the January 27th edition of Science.
The human genome has at least 20,000 individual genes, and from each gene, proteins are processed into various forms (or "proteoforms"). So, for the 20,300 genes, there are millions of unique proteoforms created due to genetic variation, modification or alternative splicing. In order to understand the functioning of a biological system, one must know the nature, localization and abundances of these proteoforms in addition to how they interact with each other.
The project titled the "Blood Proteoform Atlas" (BPA) studies a collection of the primary structures of around 30,000 unique proteoforms expressed from 1,690 human genes across 21 cell types and plasma from human blood and bone marrow.
Speaking to Bioworld Science, senior author Neil Kelleher said that the BPA was currently "the largest study on proteins and can be used to understand the spatial and temporal dynamics of how proteins function in human tissue. Proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types."
Kelleher is the Walter and Mary Glass Professor of Molecular Biosciences and professor of chemistry in Northwestern's Weinberg College of Arts and Sciences and director of the Chemistry of Life Processes Institute (CLP) that develops novel platforms for drug discovery and diagnostics.
The authors used a state-of-the-art mass spectrometry and data analysis technique to identify proteofoms in cells and blood efficiently, ensuring the proteoforms were intact in a form of "top-down" analysis thus avoiding the problem of inferring proteins using peptide data from shotgun proteomics analysis.
"The reason that proteins have not been very precise biomarkers is because we have been using an imprecise approach to study them," Kelleher said. "This study shows the extent to which a single protein form is observed in just one cell type and our analyses prove that proteoforms are better markers of a cell type than monitoring gene expression using just protein-level assignments."
Kelleher further added that, "we expect that proteins such as transcription factors could have more proteoforms per protein. We estimate the number of proteoforms to be approximately 1.1 million in a human cell type. Our study probably accounts for at most 3% of human proteoforms... so we need to improve technologies for systematic proteoform discovery."
Kelleher stressed that it was important that there was a biologically relevant example to contextualize how these proteoform panels can identify diseases noninvasively as markers. To show the atlas' clinical potential, the researchers used it in the study to identify cell and proteoform signatures that distinguish normal liver transplant function from acute rejection and other causes of graft dysfunction. Physicians must suppress the immune system with drug therapy and monitor liver transplant recipients for signs of rejection, often only responding after an episode has begun. With the BPA as a reference map, the team took blood samples from human patients and they then examined which proteoforms were activated in response to the transplant.
Next, the team developed a panel of 24 proteoforms from the initial study and looked at them in transplant recipient samples from across the country. They found the same proteoforms were activated as in the first trial. This can "identify patients who have no signs of rejection versus those who have very early evidence of rejection," and "if we can pick up on this several weeks before rejection actually happens, we might be able to modify immunosuppression."
"This cellular and molecular specificity can help advance the future of protein-level diagnostics and broader goals for understanding human biology," Kelleher said.
The team continues to examine how proteoforms change in transplant recipients over time to develop additional biomarkers that may facilitate better therapeutic management down the line. Kelleher said as the number of cell types in the atlas grows, so too will potential ways to use it. In addition to broadening understandings of human biology, the BPA could have similar applications across immune disorders.