Last updated: 17 Sept at 12am PST
Cells regulate protein functions in a wide variety of ways, including by
modifying the protein structure. In an instant, a protein can take on another
form and perform no or even the "wrong" function: in humans, proteins
that fold wrongly can cause serious diseases such as Alzheimer's, Parkinson's
or cystic fibrosis. Some of these proteins
also have a tendency to "infect" other molecules of the same type and
congregate into insoluble so-called amyloid fibrils or plaques. These amyloids
can damage cells and tissues and make people ill.
Method breaks the shackles
Until now, there has been a lack of methods that enable structurally
modified proteins to be recorded quantitatively in complex biological samples.
Although there is a series of techniques to study structurally modified
proteins, such as x-ray crystallography, nuclear magnetic resonance
spectroscopy and other spectroscopic techniques, they cannot be used to analyse
complex biological samples. Other procedures that researchers have used to
study structural changes ofproteins in cells also have their limits: prior to
the analysis, the proteins of interest have to be specifically marked to enable
the scientists to observe them in samples. However, this approach is only
possible for a few proteins in a sample.
The team headed by Paola Picotti, a professor of protein network biology,
has now found a way to measure the majority of structurally modified proteins
in any biological sample, which can contain thousands of different proteins.
Picotti and her team have succeeded in measuring quantities of structurally
modified proteins directly from a complex protein mixture as it occurs in cells,
without cleaning or enriching the samples.
Combination of several methods
For their new method, the researchers combined an "old"
technique and a modern approach from proteome research. First of all, familiar
old digestive enzymes such as proteinase K are added to the sample, which cut
the proteins depending on their structure into smaller pieces known as
peptides. The fragments can then be measured using a technique which Picotti
played a key role in co-developing during her time as a postdoc at ETH Zurich
(as ETH Life reported). Known as Selected Reaction Monitoring (SRM), this
method enables many different peptides to be sought specifically and their
quantities measured. Based on the peptides found, proteins that were originally
present in the sample can be determined and quantified.
What makes it so special: the digestive enzymes cut the same kind of
proteins that have different structures in different places, resulting in
diverse fragments. Like a fingerprint, these fragments can be clearly assigned
to the individual structures of the protein.
"This means we can use the method to analyse structural changes of
specific proteins or entire protein networks in a targeted fashion and measure
numerous proteins at the same time," says Picotti.
Works for protein responsible for Parkinson's
Based on their new method, the researchers devised a test to specifically
measure the "healthy" and "sick" versions of the protein
alpha-synuclein in complex, unpurified samples such as blood or cerebrospinal
fluid. Alpha-synuclein is thought to cause Parkinson's when its structure is
modified. The pathological structural variety congregates with its own kind to
form amyloid fibrils, which harm neuronal cells.
With the aid of the test, the scientists managed to measure the exact
amount of pathogenic and non-pathogenic alpha-synuclein directly in a complex
sample. The test also yielded information on the structure of the protein.
"It shows us which parts of the protein change and turn into the new
pathological structure," says Picotti.
Increasing number of amyloidoses
For the time being, the concentration of alpha-synuclein cannot be used
as a biomarker as the levels of the protein are too similar in the blood or
cerebrospinal fluid of Parkinson's sufferers and healthy people. "Nevertheless,
it is possible that the ratio of pathological versus nonpathogenic
alpha-synuclein structure changes with time, along the progression of the
disease" suspects the ETH-Zurich professor. "As the new method
enables us to measure both structures of the alpha-synuclein protein in a large
variety of samples, it might be possible to use this to develop new biomarkers
for this disease in the future," she hopes. Using the method, it might
also be conceivable to discover other, as yet unknown amyloid-forming proteins
that are connected to diseases without prior knowledge.
Both applications - the quantification of a specific known protein with a
modified structure and the discovery of new proteins with variant structures -
are highly relevant from a medicinal perspective, Picotti explains. "The
number of amyloidoses, i.e. diseases that develop due to changes in protein
structures, increases every year."
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