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Proteomics Method Detects Changes in Protein Profile for Mad-Cow Disease

Researchers from Canada and Germany have demonstrated that proteomic methods may be able to distinguish cattle infected with bovine spongiform encephalopathy from healthy ones, opening up the possibility that a test could be developed in the future that would allow live animals to be tested for the untreatable and deadly ailment.
In the two decades since BSE, commonly called mad-cow disease, was first described, sporadic outbreaks in the UK, Canada, and elsewhere have occurred leading to mass slaughters of cows feared to be infected, and large-scale recalls of beef.
The disease is also believed to be transmittable to humans — through consumption of BSE-infected products — in the form of Creutzfeldt-Jakob disease, identified in 1996. So far, about 200 people worldwide have died from the disease, though the figure is expected to rise due to the disease’s incubation period of about four years. 
While a protein is known to be associated with BSE, a disease-related isoform of the prion protein, PrPd, the only way to detect PrPd is through an autopsy. But using two-dimensional differential gel electrophoresis and mass spectrometry, the team of 10 researchers was able to detect changes in the protein profile of the urine of cows infected with the disease.
Described in an article published Sept. 5 in the online edition of the journal Proteome Science, the work could pave the way for new ways of testing for BSE.
David Knox, the corresponding author on the study, and a researcher at the Public Health Agency of Canada’s National Microbiology Laboratory, was not available for comment but in a statement said, “We are hopeful that the knowledge that we’ve gained from this study will eventually lead to a live test.”
Knox and his colleagues’ work also identified a subset of proteins that may be able to determine how far BSE has progressed.
“Our work shows that it is possible to identify biomarkers in urine that could be useful in the diagnosis and monitoring of disease progression in BSE and related transmissible spongiform encephalopathies,” Knox added.
A test that could be used on live livestock would be of particular importance for assessing the health of breeding stock, for which post-mortem testing is not practical, the researchers said in the article.
Work on such an assay has been in development but have been complicated by the “extremely” low levels of PrPd present in accessible tissues and fluids, such as blood, urine, and cerebrospinal fluid, according to Knox and his co-researchers.
“Furthermore, the demonstration that most infectivity is associated with protease sensitive forms of PrPd also calls into question the reliability of tests reliant on the association of prion infectivity with the presence of a proteinase K resistant fragment that is measured by Western blotting, enzyme-linked immunosorbent assay, or immunohistochemistry,” they added.
A test called the protein misfolding cyclic amplification assay offered some promise as a live-test alternative. PMCA assays, using relatively defined components, have been able to detect PrPd in CSF, serum, and urine from terminal-stage hamsters, but, the authors said, additional investigation and validation of such methods are needed.
“Thus the identification of alternative biomarkers in accessible tissues or body fluids applicable to the development of diagnostic tests remains a relevant approach,” the authors said.
Testing Without Slaughtering
For their work, Knox and his co-researchers chose urine because of its easy collection and less-complex protein profile. Their work, they said, builds on earlier research implicating the presence of protease-resistant light-chain immunoglobulin in urine as surrogate marker for prion diseases.
To arrive at their results, the researchers tested four cows orally infected with BSE and four age-matched controls. Urine samples were collected from each cow and labeled with CyDye. The resulting gels were then analyzed and spot features of interest were excised and prepared for protein digestion.

“Our work shows that it is possible to identify biomarkers in urine that could be useful in the diagnosis and monitoring of disease progression in BSE and related transmissible spongiform encephalopathies,”

They then used an Agilent 1100 system to perform nanoflow LC of tryptic peptide samples, and eluted peptides directly onto an Applied Biosystem QStar XL hybrid LC-MS/MS system. MS/MS data were acquired for the entire LC run, and the data was searched against an National Center for Biotechnology Information database using Mascot version 2.2.
Gel images were analyzed with the DeCyder Differential In-gel Analysis module with version 6.5 of a GE Healthcare DeCyder 2D software, and DIA files were imported into the Biological Variation Analysis module. A total of 1,329 mast-spot features were detected, quantified, and matched.
A total of 27 gels were run to obtain gel images suitable for analyses, resulting in 46 gel samples representing 46 biological samples and 24 gel images of an internal standard created by pooling equivalent amounts of protein from each urine sample.
Gel images were grouped so that samples from each cow formed a group, resulting in eight groups each representing one of eight biological replicates. The data were then filtered so that only the 36 spot features showing statistically significant changes in abundance and present on all 46 gel images were considered for ensuing analyses.
Principal-component analysis was performed on the data set “to identify the relative contributions of the inherent differences between individuals and disease state on the variance exhibited by the eight biological replicates,” according to the authors. The analysis demonstrated that the cows generally segregated into infected and healthy groups “indicating that the disease status of the animals was the primary factor affecting the differential abundance of urinary proteins,” they added.
Further PCA analysis was performed on gel images that were grouped according to disease state and the length of time that cows were infected. The cows again segregated into infected and control groups, further indicating that the disease status of the animals was the primary cause of the differential abundance of urinary proteins observed.
In addition, the researchers observed that in the data set of infected cows, data points clustered together based on the length of time that cows were infected and generally moved in a specific direction as disease progressed. Healthy samples showed a similar pattern, though it was less pronounced.
According to the authors, this suggests that progression of the disease factored into the differential abundance of urinary proteins observed.
“Significantly, the markers of disease progression demonstrated very little overlap with those identified as able to track age,” the researchers said. “This indicates that they were a measure of disease specific progression and that their identification may also provide insight into the pathology of these diseases.”
Smoking-Gun Protein?
Further analyzing the samples with LC-MS/MS, the researchers detected five proteins at elevated levels in the urine of infected cows: clusterin, immunoglobulin gamma-2 chain C region, cystatin, cathelicidin 1, and GCAP-11/uroguanylin. The roles that these proteins play in BSE, if any, is unclear, the researchers said.
But the researchers suggest that two of the proteins, immunoglobulin gamma-2 chain C region and clusterin, may have potential as biomarkers for the disease.
They wrote that although the increased abundance of immunoglobulin in urine associated with BSE “was probably due to a change in immunoglobulin concentration in the blood plasma … the detection of differential abundance of another immunoglobulin protein in urine by an unbiased screen lends support to the suggestion that immunoglobulin light chain may constitute a surrogate marker for” transmissible spongiform encephalopathy, of which BSE is a type.
Meantime, they wrote that clusterin was able to distinguish infected cows from healthy ones all of the time throughout the experiment, but added that increased levels of the protein have also been observed in models of other neurodegenerative diseases, such as Alzheimer’s disease, as well as a number of renal insults, raising questions about the specificity of clusterin as a biomarker of BSE in cattle.
“The specificity of the particular isoform of clusterin observed to best discriminate BSE infected and control cattle remains to be seen,” they said.
The authors said the results of their study demonstrate that, in principle, biomarkers for TSE diseases, including BSE, can be identified by analyzing changes in the urine protein profile “provoked by the disease.” In future work, a larger sample size that also includes cattle of different strains will be needed to test the value of the biomarkers identified, they said.

A variety of pre-fractionation methods will also be used in future studies to overcome the bias of 2D-DIGE in identifying abundant proteins, resulting in other potential biomarkers that may have been missed.

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