以人工培养的中枢神经系统血管母细胞瘤(Central nervous system hem angioblastoma,HB)细胞为研究对象,发展了蛋白质组学分析方法,鉴定了HB细胞与人脑神经元细胞的差异蛋白质。采用在线HPLC串联LTQ-Orbitrap质谱鉴定样品的可溶性蛋白质,得到了HB细胞的蛋白质组表达谱。HB细胞鉴定得到674个蛋白质,神经元细胞鉴定获得531个蛋白质。根据基于肽段鉴定的蛋白质组半定量分析方法对质谱数据进行蛋白质的差异比较分析,发现了波形蛋白(Vimentin),14-3-3epsilon蛋白和碳酸酐酶Ⅱ(Carbonic anhydraseⅡ,CAⅡ)等在HB细胞中表达量发生明显变化的蛋白质,并对其进行了免疫组织化学染色分析。结果显示,波形蛋白(Vimentin)、14-3-3epsilon蛋白以及碳酸酐酶Ⅱ(CAⅡ)等蛋白质表达量的改变与HB的发病密切相关,对探索HB的起源有重要意义。
The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3-10 and 4-7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5-9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.
MI WeiLIU XinJIA WeiLI LeiCAI YunYING WanTaoQIAN XiaoHong
By means of the reaction between a DOTA-NHS-ester bifunctional reagent and N-terminal peptides of proteins, and then che- lation of lanthanide metal ions as tags, we established a novel method for the identification of N-terminal peptides of proteins and their relative quantification using metal-element-chelated tags coupled with mass spectrometry. The experimental results indicate that metal elements are able to completely label N-terminal peptides at the protein level. The N-terminal peptides are enriched as the peptides digested with trypsin are selectively eliminated by isothiocyanate-coupled silica beads. We success- fully identified the N-terminal peptides of 158 proteins of Thermoanaerobacter tengcongensis incubated at 55 and 75 ℃, among which N-terminal peptides of 24 proteins are partially acetylated. Moreover, metal-element tags with high molecule weights make it convenient for N-terminal peptides consisting of less than 6 amino acids to be identified; these make up 55 percent of the identified proteins. Finally, we developed a general approach for the relative quantification of proteins based on N-terminal peptides. We adopted lysozyme and ribonuclease B as model proteins; the correlation coefficients （R2） of the standard curves for the quantitative method were 0.9994 and 0.9997, respectively, with each concentration ratio ranging from 0.1 to 10 and both relative standard derivations （RSD） measured at less than 5%. In T. tengcongensis at two incubation tem- peratures, 80 proteins possess quantitative information. In addition, compared with the proteins of T. tengcongensis incubated at 55 ℃, in T. tengcongensis incubated at 75 ℃, 7 proteins upregulate whereas 16 proteins downregulate, and most differential proteins are related to protein synthesis.
