Modified Protein Structures Analyzed by Selected Reaction Monitoring

A novel method has been developed to measure the majority of structurally modified proteins in any biological sample, which can contain thousands of different proteins.

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.


Scientists at the Swiss Federal Institute of Technology, (ETH; Zurich, Switzerland) combined an older technique and a modern approach from proteome studies. The established digestive enzymes such as proteinase K were 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 called 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.

The team used various techniques in their study where aliquots of the sample were subjected to the thioflavin T (ThT) binding assay, to circular dichroism, and Fourier transform infrared spectroscopy measurements and were imaged by transmission electron microscopy (TEM) to confirm the formation of amyloid-like fibrils. For the ThT assay fluorescence emissions were recorded at 484 nm on a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies Inc.; Loveland, CO, USA). Fourier transform infrared spectroscopy measurements were conducted on a 1,720× spectrometer (PerkinElmer Life Sciences; Waltham, MA, USA).

The investigators devised a test to specifically measure the normal and altered 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.

Paola Picotti, PhD, a professor of protein network biology and senior author of the study, said, “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. The number of amyloidosis, diseases that develop due to changes in protein structures, increases every year.” The study was published on September 14, 2014, in the journal Nature Biotechnology.

Related Links:
Swiss Federal Institute of Technology
Agilent Technologies
PerkinElmer Life Sciences