Researchers from Switzerland-based ETH Zurich and the University of Padua in Italy say they have found a way to measure the majority of structurally modified proteins in any biological sample, which can contain thousands of different proteins. They add that measurements can be made from complex protein mixtures such as those that occur in cells, without cleaning or enriching the samples.
The researchers published their study (“Global analysis of protein structural changes in complex proteomes”) in Nature Biotechnology.
For their new method, the scientists combined an “old” technique and a modern approach from proteome research. First, 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 known as Selected Reaction Monitoring (SRM). This method enables many different peptides to be specifically found out and their quantities measured. Based on the peptides found, proteins that were originally present in the sample can be determined and quantified.
Until now, there has been a lack of methods that enable structurally modified proteins to be recorded quantitatively in complex biological samples, according to the researchers, who pointed out that 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 analyze complex biological samples.
Other procedures that researchers have used to study structural changes of proteins 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.
What makes the new method so useful is that the digestive enzymes cut the same kind of proteins that have different structures in different places, resulting in diverse fragments, explained Paola Picotti, Ph.D., a professor of protein network biology at ETH Zurich and research team leader. Like a fingerprint, these fragments can be clearly assigned to the individual structures of the protein.
“Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ~300 proteins upon a change of nutrients,” wrote the investigators. “We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.”
“This means we can use the method to analyze structural changes of specific proteins or entire protein networks in a targeted fashion and measure numerous proteins at the same time,” continued Dr. Picotti.
Based on their new technique, 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 nonpathogenic 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,” noted Dr. Picotti.
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,” said Dr. Picotti. “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.”
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, she said.