Proteome Analysis for Early Diagnosis of Prion Disease

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Purpose :To establish the strategy for early diagnosis of prion disease, identify marker molecules associated with disease progression.Methods :A disease form of prion protein (PrP) was infected to PrP-overexpressing mouse neuron-derived cell line N2a58 or cerebral ventricles of ddY mice, and serum-free culture medium from the cells or blood samples were recovered, respectively. After concentration of the culture media by TCA precipitation and removal of serum albumin and immunoglobulin from the cleared blood sera, each sample was analyzed by 2-dimensional gel electrophoresis to identify protein spots with infection-associated behavior. Tryptic peptides were recovered from candidate spots and subjected to LC-tandem mass spectroscopy (LC/MS/MS) for protein identification.Results and Discussion :A set of protein spots of approx. MW 60,000 with pI 4-5 and MW 14,000 with approx. pI 7.0 was increased in the culture media from disease form PrP-infected cells. These proteins. were identified as serum proteins from bovine, which had been included in culture medium. The intense recovery of serum proteins was reproducible even after the bovine-serum containing medium was extensively washed before sample preparations. Thus, the results suggest that PrP-infection might cause some changes in cell membranes that increased the affinity to serum proteins.In the serum samples from PrP-infected mice, a protein spot found in uninfected control, mice was split into two and then gradually disappeared on the disease progression. LC/MS analysis indicated that a blood coagulation factor was involved in these spots. Physiological significance of these observations should await further studies.