Proteome Analysis for Early Diagnosis of Prion Disease

目的：建立朊病毒病的早期诊断策略，筛选与疾病进展相关的标志分子方法：将朊病毒蛋白（PrP）感染过表达的小鼠神经元源性细胞系N2 a58或ddY小鼠脑室，回收细胞无血清培养液或血样。通过TCA沉淀浓缩培养基并从澄清的血清中除去血清白蛋白和免疫球蛋白后，通过二维凝胶电泳分析每份样品，以鉴定具有感染相关行为的蛋白质点。从候选点回收胰蛋白酶肽，并进行LC-串联质谱（LC/MS/MS）用于蛋白质鉴定。MW 60，000，pI 4-5，MW 14，000，约在来自疾病形式PrP感染细胞的培养基中，pI 7.0增加。这些蛋白质。鉴定为牛血清蛋白，其已包含在培养基中。即使在样品制备前充分洗涤含牛血清的培养基后，血清蛋白的强烈回收率也是可重现的。因此，结果表明，PrP感染可能引起细胞膜的一些变化，增加了对血清蛋白的亲和力。在PrP感染小鼠的血清样品中，未感染对照组中发现的蛋白斑点，小鼠分裂为两个，然后随着疾病的进展逐渐消失。LC/MS分析表明这些斑点中涉及凝血因子。这些观察结果的生理意义有待进一步研究。

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.