Introduction. Different techniques with high diagnostic sensitivity (eg. PMCA, RT-QUIC, Exosomes) have been developed in recent years in order to make possible an ante-mortem diagnosis of prion diseases, currently based on the post-mortem detection of the pathological prion protein in the nervous system. At the same time, proteomic studies have been performed to identify alternative biomarkers in accessible tissues or body fluids of affected animals. With this purpose we studied plasma of sheep with natural scrapie by two-dimensional gel electrophoresis (2D-PAGE ). Materials and methods. Ovine plasma samples were collected from four different Italian naturally scrapie-affected flocks. Twenty animals were chosen, ten positive and ten negative sheep, coupled by age, sex and breed. To increase the visibility of low abundant proteins, plasma samples were subjected to ProteoMiner(TM) (Biorad) before 2D-PAGE. Spots were detected by colloidal Comassie blue staining; image analysis and gel comparison were carried out by BioNumerics ® software; proteins were identified by nanoLC-ESI-LIT-MS/MS (Thermo) analysis and database search by MASCOT software package (Matrix Sciex). Results. Two spots, with isoelectric point between 6.5 and 7 and molecular weight between 65 and 70 kDa, have shown a statistically significant increased expression in the positive scrapie samples. NanoLC-ESI-LIT-MS/MS analysis has been carried out on these spots, which have been identified as isoforms of fibrinogen ? chain. Conclusion. Fibrinogen is a multimeric protein, synthesized by liver, which undergoes several post-translational modification, such as hydroxylation, glycosylation, phosphorylation and sulphation. Anyway plasma fibrinogen concentration is conditioned by a great number of physiological and pathological stimuli, so in this study we investigated the possible presence on fibrinogen ? chain of specific post-translational modifications in scrapie affected animals. To assess the specificity of this potential marker, further analyses are undergoing in our laboratory on plasma samples from sheep with different pathologies.
Fibrinogen beta chain as potential biomarker for an intra-vitam scrapie diagnosis
D'Ambrosio Chiara;Scaloni Andrea;
2014
Abstract
Introduction. Different techniques with high diagnostic sensitivity (eg. PMCA, RT-QUIC, Exosomes) have been developed in recent years in order to make possible an ante-mortem diagnosis of prion diseases, currently based on the post-mortem detection of the pathological prion protein in the nervous system. At the same time, proteomic studies have been performed to identify alternative biomarkers in accessible tissues or body fluids of affected animals. With this purpose we studied plasma of sheep with natural scrapie by two-dimensional gel electrophoresis (2D-PAGE ). Materials and methods. Ovine plasma samples were collected from four different Italian naturally scrapie-affected flocks. Twenty animals were chosen, ten positive and ten negative sheep, coupled by age, sex and breed. To increase the visibility of low abundant proteins, plasma samples were subjected to ProteoMiner(TM) (Biorad) before 2D-PAGE. Spots were detected by colloidal Comassie blue staining; image analysis and gel comparison were carried out by BioNumerics ® software; proteins were identified by nanoLC-ESI-LIT-MS/MS (Thermo) analysis and database search by MASCOT software package (Matrix Sciex). Results. Two spots, with isoelectric point between 6.5 and 7 and molecular weight between 65 and 70 kDa, have shown a statistically significant increased expression in the positive scrapie samples. NanoLC-ESI-LIT-MS/MS analysis has been carried out on these spots, which have been identified as isoforms of fibrinogen ? chain. Conclusion. Fibrinogen is a multimeric protein, synthesized by liver, which undergoes several post-translational modification, such as hydroxylation, glycosylation, phosphorylation and sulphation. Anyway plasma fibrinogen concentration is conditioned by a great number of physiological and pathological stimuli, so in this study we investigated the possible presence on fibrinogen ? chain of specific post-translational modifications in scrapie affected animals. To assess the specificity of this potential marker, further analyses are undergoing in our laboratory on plasma samples from sheep with different pathologies.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
