A method that facilitates the discovery of biomarkers for psychiatric and immune-related diseases

Brazilian researchers show that proteins associated with diseases and key biological processes can be found in the blood plasma fraction usually discarded in proteomic analysis.

By André Julião  |  Agência FAPESP – Analysis of all the proteins present in a blood plasma sample can reveal the occurrence of a number of processes in the organism and even help diagnose certain diseases.

This type of study is performed using a small part of any sample: only 14 of the most abundant proteins represent 90% of the total protein mass in human plasma, while thousands of different proteins make up the remaining 10%. Some of these proteins are used as markers of biological processes.

Therefore, before performing a proteomic analysis, researchers chemically separate the more abundant proteins from those that are less plentiful in plasma. This process is known as depletion.

However, scientists at the University of Campinas (UNICAMP) in the state of São Paulo, Brazil, have shown that this separation is not as precise as is often thought and can leave important proteins out of the analysis. The study was part of a research project supported by FAPESP.

When they investigated the most abundant proteins in plasma, which are usually discarded, the researchers observed that such proteins were bound to other, less abundant proteins believed to be present in the portion used in the analysis. The study showed that some of these normally excluded proteins regulate important biological processes and could serve as markers of disease.

The findings are reported in the journal Separation Science Plus.

“The primary aim of the study was to alert the scientific community to this problem. Researchers may literally be discarding potential biomarkers, and this may have influenced the findings of previous studies. We demonstrate that some of the proteins excluded from the analyses performed to date are potential biomarkers of several conditions,” Daniel Martins-de-Souza, a professor at the University of Campinas’s Biology Institute (IB-UNICAMP) and principal investigator for the study, told Agência FAPESP.

The investigation was conducted during the master’s research of Licia Carla da Silva Costa, first author of the article, as part of a project dedicated to identifying biomarkers of schizophrenia by means of proteomics, which is the analysis of the entire set of proteins in an organism or biological sample.

“Our lab focuses on investigating biomarkers of mental diseases. The main finding of this study is that the fraction of abundant proteins should not be discarded in this kind of analysis,” Costa said.

From Cambridge to Campinas

In partnership with Johann Steiner, a professor of psychiatry at the University of Magdeburg in Germany, the researchers at UNICAMP created an approach they call the “depletome”, which entails analyzing the proteins hitherto discarded in the depletion stage of plasma proteomic analysis.

The idea arose during Martins-de-Souza’s postdoctoral internship at the University of Cambridge (UK) between 2010 and 2012.

Analyses with the techniques available at the time showed that some of the less abundant proteins were being discarded. The study was resumed only in 2016, during Costa’s scientific initiation project.

The new analyses used a high-resolution mass spectrometry technique known as shotgun proteomics, which is based on digesting proteins into peptides and their building blocks.

The researchers analyzed plasma samples from 20 healthy individuals and identified 12,714 peptides corresponding to 281 proteins. After grouping proteins in the same family and hiding those with peptides that were a subset of another protein’s peptides or had few corresponding peptides in the plasma, 198 proteins remained.

A search of protein function databases found 35 biological processes associated with the 198 proteins. Receptor-mediated endocytosis was the process with the highest number of associated proteins (19).

“Endocytosis is a process whereby the cell takes up macromolecules and particles from the surrounding medium,” Martins-de-Souza explained. Alterations in this pathway, he added, may be associated with the onset of psychiatric and immune system disorders, as well as some types of cancer. Endocytosis can also be leveraged in precision medicine for customized treatment and drug delivery.

Some 20 proteins involved in the immune response were found in the depletome, making this fraction a source of possible biomarkers for metabolic, neurodegenerative and psychiatric diseases.

The important biological processes associated with this group of proteins also included oxygen transport to cells, vasodilation and blood coagulation, among others.

The researchers believe other proteins can be found in addition to the 198 described in the article. “The next step in this research is to refine the method and obtain more potential biomarkers,” Martins-de-Souza said.

The article “Blood plasma high abundant protein depletion unintentionally carries over 100 proteins” (doi: 10.1002/sscp.201900057) by Licia C. Silva-Costa, Sheila Garcia-Rosa, Bradley J. Smith, Paulo A. Baldasso, Johann Steiner and Daniel Martins-de-Souza can be retrieved from onlinelibrary.wiley.com/doi/full/10.1002/sscp.201900057.