Researchers have developed a groundbreaking technology that enables them to measure how proteins are broken down and built up in single human cells – a discovery with great potential for personalised medicine and improving treatment for people with conditions such as dementia, cancer and infertility.
When the brain is affected by dementia, the proteins that keep our brain cells functioning slowly and nearly invisibly break down. But how and when this happens and what causes certain cells to continue to function or break down have long been unknown.
Researchers in Denmark and other countries have now developed a pioneering method that enables them to determine the activity of proteins in individual cells. This could provide important knowledge about dementia, cancer and other disorders in which cells change their activity and thereby lead to much more targeted treatments than those available today.
“We could previously only measure how much protein is present in each cell. Now we have developed a method to determine how proteins are produced and broken down,” explains lead author Jesper Velgaard Olsen, Professor and Executive Director, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark. “We can now determine which proteins are active in which cells – and how they change over time.”
The body’s service workers
Understanding why being able to measure proteins is so important requires knowing their role in the human body. Our genes, the DNA, provide the recipe – but the proteins do the work. They ensure that cells divide, that energy is converted and that we can think, move and fight infections.
Every cell in the body contains thousands of proteins that are constantly being produced, used and broken down. But researchers had only been able to determine how much of each protein was present in the cells – not their activity and certainly not in individual cells. The new method has changed this.
“This is equivalent of seeing a still image to following a film and determining what is actually happening inside the cells,” says Jesper Velgaard Olsen.
This single-cell proteomics technology enables researchers to study the proteins in a single cell and not just in large groups of cells, as was done previously.
This is important because cells can act very differently even though they initially look quite similar. Researchers can determine which proteins are active in each cell and how they function. This can reveal details that would otherwise be missed when examining many cells together.
Instead of getting an average of many cells, they can get a very precise picture of what is happening inside each individual cell – and this can help to understand diseases such as cancer, in which some cells act differently than others.
A molecular detector
One of the researchers’ most important tools is mass spectrometry, which separates charged molecules in an electric or magnetic field according to their mass and charge.
The result is a spectrum – a kind of molecular fingerprint – that researchers can use to identify precisely which molecules are present and how many in the material they are examining.
Mass spectrometry can reveal which proteins are present in a cell and in what quantities, providing very detailed understanding of the cell’s content and activity.
With the right equipment, researchers can measure thousands of proteins in single cells. By labelling the cells in a specific way, researchers can monitor which proteins are new, which are old and how quickly they are replaced. This provides new insight into the activity of proteins and their importance for the individual cell – including how actively the proteins turn over in an individual cell.
To monitor the turnover, the researchers use two versions of the same amino acid – the building blocks of protein. This amino acid differ in the carbon isotopes, with one built around the common carbon-12 atom and the other based on the heavier carbon-13 atom.
“The same protein can act very differently depending on the type of cell, such as whether a cancer cell is dividing or dormant. The turnover of a protein in a stomach cell can differ greatly. This can tell us how active the protein is and how important it is for certain types of cells,” notes Jesper Velgaard Olsen.
From cancer to stem cells
The researchers used the method in two types of experiments. In one, they monitored how the activity of the proteins changes when stem cells develop into specialised types of cells such as nerve cells or cardiac muscle cells. Some identical proteins become very active in cardiac muscle cells but are almost inactive in nerve cells.
In the other experiment, the researchers used the method to distinguish between cancer cells that are dividing and those that are dormant. This could lead to better cancer treatment, since many types of chemotherapy attack cells that divide rapidly. Identifying which cancer cells are active and which are just lying around may enable treatment to be adapted more precisely in the future.
Jesper Velgaard Olsen says that histones are another type of protein that could be useful to study with the new technology. Histones function as small coils around which the DNA strands are wound inside the cell nucleus to take up less space.
In addition to acting as packaging material, histones also regulate which genes turn on or off. They are therefore crucial in determining which other proteins the cell produces and how it acts in general.
Isotope labelling shows how histones reveal whether a cell is actively dividing or dormant. Cells without recent histone build-up do not divide, whereas those with new histones are dividing.
“The technology suddenly enables us to distinguish between cancer cells that are growing and those that are dormant,” observes Jesper Velgaard Olsen.
On the edge of the possible
The researchers hope that the new method can eventually be used to design more targeted medicine. Instead of giving the same medicine to everyone with the same diagnosis, researchers can study how each person’s cells respond – and adapt the treatment accordingly. This is personalised medicine, and the potential is enormous.
Jesper Velgaard Olsen emphasises, however, that the technology is still being developed.
“This is bleeding edge technology – we are at the forefront of what can be measured. Hospitals cannot start using it tomorrow, but we are already using it in our research laboratories to answer important biological questions,” he says.
The study is the result of many years of work to improve both the technology and the biological models. Protein turnover in single cells could not be measured until now, because the cells are so small and because the proteins are constantly changing. But by combining the isotope-labelled amino acid method with extremely precise mass spectrometry, the researchers have gotten very close to the internal chemistry of the cells – and can monitor this in real time.
Egg cells are strange
One next step is to use the method to study the very first budding phases of life. The researchers want to determine how proteins are broken down and rebuilt in the earliest stages after fertilisation – when a human egg begins to divide into two, four, eight cells. This could provide knowledge about heredity, diseases and perhaps also fertility problems.
On that front, there has been some controversy about understanding what is actually happening, says Jesper Velgaard Olsen. He reports that researchers in the field have sharply disagreed about how much protein synthesis and turnover occurs during the early stages of development up to the first cell divisions. The answer is not obvious, since the egg is extremely large compared with the cells that ultimately end up in the human body.
In the beginning, when the fertilized egg cell divides into two, four, eight cells, the overall size does not change, remaining fairly intact. But the cells get smaller and smaller as they divide. Some of the proteins are specifically degraded, and others are synthesised in this process.
“Perhaps our new technology can help to find answers and thus help childless parents,” says Jesper Velgaard Olsen.
He emphasises that there is still a long way to go before single-cell proteomics becomes standard in hospitals. But the path has been laid. Researchers have accessed the hidden life of proteins, which could strongly affect how we understand diseases and treat the people who have them in the future, he states.
Second time lucky
Jesper Velgaard Olsen started his career as an analytical chemist, working with mass spectrometry and analysing molecules. There he discovered that the same method could be used to determine proteins in cells, which ignited his curiosity. What really captured him was the opportunity to understand how proteins change and function in the human body – a secret world opened up through technology.
Jesper’s work with proteomics pioneer Matthias Mann in his biotech company in the early 2000s was a seminal experience, with ambitions to find disease markers through mass spectrometry. Although the company did not last, it gave Jesper invaluable experience and set the course for his further research, when he returned to academia, this time as a member of Matthias Mann’s group, to perform research more deeply into the role of proteins in diseases.
Now, with the major breakthrough, Jesper can conclude that the timing is finally right and that the technology is ripe to make a real difference in understanding diseases and developing future medicines.
