For the first time, researchers can measure thousands of proteins inside a single cell. This offers a rare glimpse into the cell’s engine room — and shows how tiny imbalances may trigger disease long before symptoms appear.
For several years now, researchers have been able to study what happens inside individual cells.
With single-cell messenger RNA (mRNA) sequencing, researchers can determine which blueprints a cell has for building its proteins. This can be the first step towards disease – for example, when disease develops or during fetal development.
If, for example, more blueprints (pieces of mRNA) are made for some proteins, this tells researchers one thing, and more blueprints of another kind tell them something else.
However, mRNA data only show which recipes the cell has available and not whether it actually uses them to produce proteins. Many of the recipes may simply end up on the shelf without being turned into proteins.
That is why researchers have now developed a method by which they no longer just study the blueprints but measure the proteins themselves directly.
Although the technology is still in its early teenage years, it could be a breakthrough: it can deliver a picture of the inner workings of cells with unprecedented detail – and perhaps reveal what sparks disease long before symptoms appear.
“We are achieving greater detail in our studies and thereby improving understanding of what happens at the cellular level when cells function normally — and when they do not. With mRNA, we only examine the recipe – now we can finally taste the finished meal,” says a researcher behind the development of the new method, Bo Porse, Clinical Professor from the Finsen Laboratory at Rigshospitalet, Copenhagen and the Biotech Research and Innovation Centre, University of Copenhagen, Denmark.
The research has been published in Science.
A new layer of knowledge – directly from the proteins
The newly developed method is based on mass spectrometry of individual cells.
This is equivalent to weighing every building block in the cell to determine exactly which proteins it contains.
Bo Porse explains that the technology solves many of the problems associated with current methods.
“With single-cell mRNA sequencing, we can only predict which proteins a cell might make – but we cannot be sure it actually makes them. Now we can measure the proteins themselves directly. Several studies have also shown that the amount of mRNA in a cell does not necessarily reflect how many proteins are actually being produced,” he explains.
From average values to single cells
Bo Porse and colleagues will primarily use the method to understand blood cells – and what goes wrong when they become diseased.
Previously, when researchers wanted to examine protein levels in blood cells, they had to examine the blood as one large mixture of cells, from which they could only take an average measurement to say something about, for example, a person’s state of health.
Single-cell mRNA sequencing enabled researchers to examine individual cells and, for example, differentiate between what happens in red blood cells, white blood cells or other types of blood cells.
Studies of proteins sharpen this view.
Small signals with great significance
To test the method, the researchers followed how the balance between the amount of mRNA and proteins changes as immature blood stem cells slowly mature into normal blood cells.
They did this by measuring how much mRNA and how many proteins the cells contained and comparing the ratio between them to determine how it changed over time.
“Even though the correlation is good at certain times, this changes over time. For example, the quantities of mRNA and proteins are correlated well at the end of the process, but just before the stem cells decide which type of blood cell to become, the quantities of mRNA and proteins suddenly are no longer correlated, which is incredibly exciting,” says Bo Porse.
The researchers can determine that some proteins disappear without a trace from the stem cells – even though the quantity of mRNA recipes remains unchanged.
“If we only had data on mRNA, we would never have discovered this. Then we would never have realised that some of these proteins are crucial for stem cells to develop into other cell types. We have also conducted experiments with switching off these proteins and found that the stem cells lose their stem cell identity,” says Bo Porse.
Bo Porse explains that the technology for examining proteins in individual cells still needs further development, but for now it complements data about what their basic blueprints suggest.
Over time, however, Bo Porse thinks that in many cases examining the content of proteins in cells will be more relevant rather than the link between them (mRNA).
Traces of disease in the protein landscape of cells
Bo Porse’s group will use the technology to learn more about diseases of the blood.
One example is the blood disorder VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic), in which defects in the cells’ degradation system cause old or defective proteins to accumulate.
In this case, examining which proteins are actually present in the cells will be much more relevant than just what the basic blueprints say.
Something similar happens in diseases in which errors in the cell ribosomes (protein factories) cause new proteins to be built incorrectly — and therefore unable to perform their vital tasks.
Here, too, what the blueprints tell us is less relevant than examining which proteins are actually present inside the cells.
“We are preparing to conduct many studies of this type, which will give us new insight into several diseases we do not yet thoroughly understand. The method can also be used for many other diseases for which researchers have so far only had the recipes – but have not been able to determine what actually happens in disease cells,” concludes Bo Porse.
