According to a researcher, a method that can characterise the protein content of individual cells may advance knowledge about diseases and the proteins various drug candidates should target to cure them.
Understanding how an organ functions and what goes wrong when it malfunctions requires knowing what is happening at the cellular level, including proteins.
Nevertheless, organs have many types of cells, so analysing the protein content of an organ with a multitude of cell types would be like analysing a smoothie with banana, apple and orange – and trying to determine which proteins derive from which fruit.
However, researchers have developed a method to analyse the protein content of single cells, which enables them to determine how the cells in an organ differ and how diseased cells differ from healthy cells.
“The method enables us to elucidate fundamental biological aspects of the development of disease, such as how various blood diseases develop. We can now analyse whether the disease signature is present at the protein level and can thereby also identify new therapeutic targets,” explains a researcher involved in developing the method, Bo Porse, Professor, Finsen Laboratory, Center for Cancer and Organ Diseases, Rigshospitalet and University of Copenhagen.
The research behind the development has been published in Nature Communications.
Better than mRNA-based analysis
Analysing the protein expression of individual cells has traditionally focused on messenger RNA (mRNA), the blueprint from which proteins are made. This provides insight into which proteins are expressed inside the cells and in what quantities.
However, the problem with analysing mRNA as a proxy for the actual proteins is that it does not take into account breakdown of proteins or other processes that may differentiate the quantity of mRNA from the quantity of proteins.
“Many processes regulate the stability of proteins, so the cells may well contain mRNA, but some of the proteins may have been degraded. Various experiments have painstakingly extracted the quantities of mRNA and proteins from a single cell and then examined this to determine the association between the mRNA expression and the protein expression. The same experiments have also been performed on organs and show an association but not a perfect one. For some proteins, the quantity of protein correlates with the quantity of mRNA, but for others, the quantity of protein may rise despite a reduction in the quantity mRNA, or vice versa” says Bo Porse.
Fine-tuning the method to characterise proteins at the single-cell level
To get around the problem of analysing mRNA to determine the protein expression of cells, Bo Porse and colleagues developed a method to directly analyse the content of proteins in single cells.
Mass spectrometry has long been used to analyse the protein content of samples, but Bo Porse and colleagues have fine-tuned mass spectrometry and the methods for extracting proteins from individual cells.
This has enabled them to measure the quantities of 1,000 proteins in 140 cells in one day.
“This still represents fewer cells and proteins than can be analysed using mRNA, but we are constantly increasing the numbers of both cells and proteins,” explains Bo Porse.
Discovered differences in leukaemia cells
Bo Porse is not an expert in mass spectrometry but focuses on the biology and the knowledge obtained from the experiments using the method.
In the article in Nature Communications, the researchers show how the method can differentiate between different cancer cells in a cell culture established from a leukaemia patient.
Leukaemia cells are typically of three types: stem cells, progenitor cells and blast cells.
These leukaemia cells express proteins differently depending on their stage, and the researchers identified these expression patterns for the different cell stages as a proof of concept.
“We can use the method to characterise the protein expression of individual cells, and since then we have used the method on almost everything that has a pulse ,” says Bo Porse.
Discovering new drugs
Bo Porse says that being able to characterise the protein expression of individual cells has several interesting perspectives.
The researchers can now compare the mRNA expression and the protein expression in individual cells.
By characterising the relationship between mRNA and proteins at the single-cell level, the researchers can thus elucidate the fundamental molecular biological aspects of how information is translated from DNA to mRNA to proteins.
In addition, the knowledge about the protein expression in individual blood cells can probably also be used to identify new therapeutic targets for people with such diseases as leukaemia and many other organs and diseases.
Some proteins may be expressed in diseased cells but not in healthy cells, and they could be targets for new drugs.
“The method enables new therapeutic targets to be discovered. Making a new drug requires showing that the proteins that are the target of the drug are present in the cells you want to target. Our method enables us to determine this,” concludes Bo Porse.