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Catalog of protein isoforms in blood cells can predict organ rejection

The researchers discovered a blood signature of protein isoforms that could potentially predict which patients might reject a new organ transplant, helping to inform treatment decisions. The results of this study are published online in the January 27 edition of Science.

The human genome has at least 20,000 individual genes, and from each gene, proteins are made into various forms (or “proteoforms”). Thus, 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, it is necessary to know the nature, the location and the abundance of these proteoforms as well as the way in which they interact with each other.

The project titled “Blood Proteoform Atlas” (BPA) studies a collection of the primary structures of approximately 30,000 unique proteoforms expressed from 1,690 human genes in 21 cell types and plasma from human blood and bone marrow.

Talk to Science of the biological worldlead author Neil Kelleher said BPA is currently “the largest study of proteins and can be used to understand the spatial and temporal dynamics of protein function in human tissues. Proteoforms best describe biology at the level of proteins and are more specific indicators of differentiation than their corresponding proteins, which are more widely expressed in all cell types.”

Kelleher is the Walter and Mary Glass Professor of Molecular Biosciences and Professor of Chemistry at Northwestern’s Weinberg College of Arts and Sciences and Director of the Chemistry of Life Processes Institute (CLP) which develops new platforms for drug discovery and diagnosis.

The authors used a state-of-the-art mass spectrometry and data analysis technique to efficiently identify proteoforms in cells and blood, ensuring that the proteoforms were intact in a “top-down” form of analysis, avoiding thus the problem of inferring proteins using peptide data from shotgun proteomic analysis.

“The reason proteins haven’t been very accurate biomarkers is that we used an imprecise approach to study them,” Kelleher said. “This study shows the extent to which a single protein form is observed in a single cell type and our analyzes prove that proteoforms are better markers of a cell type than monitoring gene expression using only protein-level assignments.”

Kelleher added that “we expect proteins such as transcription factors to have more proteoforms per protein. We estimate the number of proteoforms at around 1.1 million in one type of human cell. Our study probably represents at most 3% of human proteoforms. …we therefore need to improve technologies for the systematic discovery of proteoforms.”

Kelleher stressed that it was important for there to be a biologically relevant example to contextualize how these proteoform panels can identify diseases non-invasively as markers. To show the clinical potential of the atlas, researchers used it in the study to identify cellular and proteoform signatures that distinguish normal liver transplant function from acute rejection and other causes of graft dysfunction. Doctors must suppress the immune system with drug treatment and monitor liver transplant recipients for signs of rejection, often not responding until after an episode begins. With BPA as a reference map, the team took blood samples from human patients and then examined which proteoforms were activated in response to the transplant.

Next, the team developed a panel of 24 proteoforms from the original study and examined them in samples from transplant recipients across the country. They found that the same proteoforms were activated as in the first trial. This can “identify patients who show no signs of rejection versus those who show very early signs of rejection” and “if we can pick this up 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 could facilitate better therapy management down the line. Kelleher said that as the number of cell types in the atlas increases, so will the potential ways to use it. In addition to expanding the understanding of human biology, BPA could have similar applications in immune disorders.