Proteome Analysis for Early Diagnosis of Prion Disease

目的：建立朊病毒疾病的早期诊断策略，识别与疾病进展相关的标志物分子。方法：将一种疾病形式的朊蛋白（PrP）感染于过表达PrP的小鼠神经元源细胞系N2a58或ddY小鼠脑室，分别从细胞和血样中回收无血清培养基。通过TCA沉淀对培养基进行浓缩，并从清除的血清中去除血清白蛋白和免疫球蛋白后，对每个样本进行二维凝胶电泳分析，以确定与感染相关行为的蛋白斑点。从候选斑点中回收色氨酸肽，用LC/MS/MS进行蛋白质鉴定。结果与讨论：一组蛋白质斑点约为。MW 6万，pI 4-5, MW 1.4万，pI约。prp感染的病源细胞培养基中pI 7.0升高。这些蛋白质。鉴定为牛血清蛋白，培养基中含有牛血清蛋白。即使在样品制备前对含有牛血清的培养基进行了大量洗涤，血清蛋白的强烈回收也可重复。因此，结果表明，prp感染可能引起细胞膜的一些变化，增加对血清蛋白的亲和力。在prp感染小鼠的血清样本中，在未感染的对照组中发现了一个蛋白质斑点，小鼠被分成两组，然后随着疾病的进展逐渐消失。LC/MS分析表明，这些斑点与凝血因子有关。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.