大鼠急性心肌梗死模型比较蛋白组学的初步研究

建立了大鼠急性心肌梗死模型, 利用二维电泳（2DE）分离正常与梗死心肌组织的蛋白, 得到不同条件下的蛋白质组图谱. 在pH 5～8范围下采用银染方法通常可以检测出超过800个蛋白点. 对比正常心肌组织, 梗死组织31个蛋白表达上调, 18个蛋白表达下调. 我们选择其中具有显著性变化的18个蛋白进行质谱分析, 并通过数据库检索, 成功鉴定了其中的8个蛋白. 其中, Hsp27, HspB6, 超氧化物歧化酶[Cu-Zn], 阿朴脂蛋白A-I, 在急性心肌梗死发病过程中起重要的作用, 可以作为临床诊断的重要标志物, 为急性心肌梗死的治疗提供药物作用靶点.     

蛋白质组学技术筛选与鉴定癫痫疾病的差异蛋白质

应用蛋白质组学双向凝胶电泳(Two-dimensional gel electrophoresis, 2DE)和质谱技术, 定量分析和鉴定了癫痫组(n=3)和正常组(n=3)脑组织的差异表达蛋白, 以从蛋白质水平上揭示癫痫病的发机制. 结果表明, 凝胶图谱可辨识2500~3000个蛋白点, 对21个显著差异表达蛋白点进行质谱鉴定和SwissProt数据库检索, 得到17个癫痫差异蛋白, 其中2个蛋白在癫痫组织中表达上调, 15个蛋白表达下调. 部分蛋白与癫痫的关系属首次报道. 这些蛋白与癫痫的发生发展相关, 可能成为癫痫的分子标志物和药物治疗的靶向蛋白.     

质谱法分析2型糖尿病大鼠神经视网膜差异表达蛋白

采用二维电泳(2DE)分离了正常SD大鼠和2型糖尿病模型大鼠神经视网膜组织总蛋白, 并用Image Master 5.0软件分析比较了正常组和糖尿病组2DE图像, 正常组检测到 3122±37(n=3)个蛋白质点; 糖尿病组检测到2702±21(n=3)个蛋白质点. 约150个蛋白质点的表达水平在两组之间存在明显差异(P<0.05). 在糖尿病组中表达上调的点68个, 下调的点82个. 选择20个差异表达蛋白质点进行肽质量指纹谱(PMF)或串联质谱鉴定, 其中7个蛋白已有报道与糖尿病视网膜病变(DR)相关, 10个蛋白尚未见有报道.     

中波紫外线与化学致癌物诱导下HaCaT细胞的蛋白表达应答

对角质形成细胞HaCaT分别进行中波紫外线(UVB)照射、2,4-二硝基苯磺酸(DNBS)刺激及UVB+DNBS(UD)共同刺激, 利用二维荧光差异胶内凝胶电泳(2D DIGE)、DeCyder定量分析软件和HPLC-nESI-MS/MS分析技术, 对HaCaT细胞产生的差异表达蛋白进行了鉴定. 有65个蛋白质点发生了明显表达差异(P<0.05), 与UVB或DNBS单独处理细胞相比, 有41个蛋白点表现为UVB和DNBS的正协同效应, 13个蛋白点表现为负协同效应, 5个蛋白点与UVB单独处理相近, 6个蛋白点与DNBS单独处理相近. HPLC-nESI-MS/MS从65个差异表达蛋白质点中共鉴定出60种单一(Unique)蛋白. 采用生物信息学方法对这些鉴定蛋白所涉及的分子功能、参与的生物学过程及信号通路进行了系统分析. 实验得到了与紫外辐射和化学诱导损伤的直接相关蛋白, 有助于研究不同环境条件下皮肤癌的形成及皮肤疾病的有效防护与治疗.     

钝顶螺旋藻Spirulina platensis两种不同形态藻丝体的光合作用和蛋白质差异表达分析

从钝顶螺旋藻Spirulina platensis中分离纯化获得了螺旋形和直线形两种不同形态的藻丝体. 通过对二者光合作用的研究发现, 螺旋形藻丝体具有较高的光饱和光合作用速率(P_m^Chla)和光饱和点, 比直线形藻丝体更能适应较高光强的环境; 而直线形藻丝体具有较低的光补偿点, 能在更低的光强下进行光合作用. 通过双向凝胶电泳对两种不同形态藻丝体的总蛋白进行比较分析, 从中找出了9个差异表达的蛋白点. 应用基质辅助激光解吸电离质谱对差异表达的蛋白质点进行鉴定和数据库检索, 结果表明, 3个蛋白与肽聚糖代谢有关, 2个蛋白与光合作用有关, 1个蛋白与细胞分裂调控相关, 1个蛋白为外膜通道蛋白, 2个蛋白为功能未知的假想蛋白.     

钝顶螺旋藻Spirulina platenis两种不同形态藻丝体的光合作用和蛋白质差异表达分析

从钝顶螺旋藻Spirulina platenis中分离纯化获得了螺旋形和直线形两种不同形态的藻丝体.通过对二者光合作用的研究发现,螺旋形藻丝体具有较高的光饱和光合作用速率(PChlam)和光饱和点,比直线形藻丝体更能适应较高光强的环境;而直线形藻丝体具有较低的光补偿点,能在更低的光强下进行光合作用.通过双向凝胶电泳对两种不同形态藻丝体的总蛋白进行比较分析,从中找出了9个差异表达的蛋白点.应用基质辅助激光解吸电离质谱对差异表达的蛋白质点进行鉴定和数据库检索,结果表明,3个蛋白与肽聚糖代谢有关,2个蛋白与光合作用有关,1个蛋白与细胞分裂调控相关,1个蛋白为外膜通道蛋白,2个蛋白为功能未知的假想蛋白.     

维拉帕米作用下急性心梗大鼠比较蛋白质组学研究

以急性心梗大鼠为研究对象, 应用双向凝胶电泳法(2-DE)分析比较了维拉帕米作用下急性心梗大鼠心肌蛋白表达的差异, 从蛋白质水平探讨了维拉帕米心肌保护作用的发生机制. 结果表明, 与假手术组及模型组相比, 维拉帕米给药组心肌组织中有8个蛋白点表达显著上调, 7个蛋白点表达显著下调. 采用质谱(MALDI-TOF-MS)分析结合数据库检索, 共鉴定了其中的15种蛋白质, 可按功能分为如下4类: (1) 能量代谢及线粒体功能相关蛋白; (2) 氧化应激相关蛋白; (3) 细胞骨架蛋白; (4) 其它蛋白. 研究结果表明, 维拉帕米的心肌保护作用与恢复心肌损伤过程中的能量供应及对抗氧化应激等作用有关.     

血管外膜肌成纤维细胞分化相关蛋白研究

为寻找涉及血管紧张素II (AngII)和转化生长因子β1 (TGF-β1)诱导的血管肌成纤维细胞(MF)分化的蛋白, 本研究采用双向电泳和质谱从整体水平检测了MF分化前后蛋白表达谱的变化, 共找到41个差异表达的蛋白点, 表达水平和/或蛋白位置在Ang II和TGF-β1刺激后都发生明显变化的蛋白点14个, 4个蛋白上调, 6个蛋白下调, 2个蛋白位置发生明显变化, 2个蛋白表达上调，位置也发生变化; 只在Ang II诱导的MF中表达发生变化的蛋白20个, 只在TGF-β1诱导的MF中表达发生变化的蛋白7个. 选取Ang II和TGF-β1共同调节的14个蛋白进行质谱鉴定, 结果除骨架蛋白外, 首次发现MF分化同泛素蛋白酶体系统和嘌呤合成有关; septin 2的下调可能是成纤维细胞分化的标志. 本研究运用蛋白质组学技术发现了新的参与MF分化的蛋白质, 为进一步研究和干预细胞表型转化提供了新的思路和靶点.     

恐惧记忆相关蛋白的蛋白质组学研究

应用双向凝胶电泳结合质谱鉴定和数据库检索, 分析比较了CD1和C57BL/6J小鼠经条件性恐惧实验后海马蛋白表达的差异, 探讨了与恐惧记忆相关的蛋白质. CD1和C57BL/6J小鼠经条件性恐惧实验后, 海马蛋白表达存在明显差异, 29种蛋白(31个蛋白点)与恐惧记忆的形成显著相关. 其中24个蛋白点表达显著上调, 7个蛋白点显著下调. 与恐惧记忆相关的蛋白按功能可分为如下6类: (1) 能量代谢或线粒体功能相关蛋白; (2) 神经发育相关蛋白; (3) 信号转导相关蛋白; (4) 细胞骨架相关蛋白; (5) 氨基酸代谢和蛋白分解相关蛋白; (6) 伴侣蛋白. 这些恐惧记忆形成的相关蛋白深化了对恐惧记忆脑机制的认识, 为研究和治疗认知相关疾病提供了新靶标.     

缺氧预处理诱导心肌细胞蛋白质组变化的初步研究

缺氧预处理(hypoxia preconditioning, HPC)可模拟缺血预处理(ischemic preconditioning, IPC)对缺血/再灌注心肌的保护作用, 涉及细胞内众多分子事件. 本工作旨在采用双向电泳和质谱分析等蛋白质组分析技术, 发现缺氧预处理后心肌细胞蛋白质整体表达上的变化, 初步分析其与缺氧预处理心肌保护作用的关系. 将原代培养的SD乳鼠心肌细胞分为2组(n＝6): (1)缺氧预处理组(HPC): 将细胞置缺氧仓内短暂缺氧20 min进行缺氧预处理(HPC), 制备心肌细胞蛋白提取物; (2)对照组(control): 细胞置于培养箱内持续常氧孵育至实验结束, 提取蛋白. 采用双向凝胶电泳和图像扫描, 经蛋白样本分离和考马斯亮蓝染色后比较分析, 选取3个差异表达蛋白点进行胶内酶切、肽质量指纹图谱分析和数据库检索. 双向电泳可分离约529±45个蛋白质, 点匹配率约为78%±7.5%. 18种蛋白质在HPC后发生明显表达差异, 其中12种蛋白质表达降低, 6种表达增高. 经质谱分析鉴定出的3种蛋白质分别为myosin light polypeptide 3, nucleoside diphosphate kinase (NDPK)和calreticulin (CRT). 缺氧预处理引起心肌细胞蛋白质组变化, 初步发现其中myosin light polypeptide 3表达下调、nucleoside diphosphate kinase和calreticulin表达增加, 可能通过调节心肌细胞的收缩性、激活G蛋白、调节细胞内Ca^2＋浓度而保护心肌. 本工作通过研究缺氧预处理延迟保护过程中心肌内源性蛋白表达水平的变化, 有助于从细胞水平探讨预处理延迟保护机制.     

Different protein expression of myocardium from Chinese mini-swine model of myocardial infarct

High-resolution two-dimensional gel electrophoresis (2-DE), followed by computer-assisted image analysis was used to screen protein patterns of normal and infarcted myocardial tissues for quantitative and qualitative differences in protein expression. In the gels of pH 5–8 immobilized pH gradient (IPG) strips, 851 protein spots were detected in normal myocardial tissue and 1 032 protein spots were resolved in infarcted myocardial tissue. Thirteen protein spots only expressed in normal myocardial tissue, and 14 protein spots only expressed in infarcted myocardial tissue. Results also showed that 49 protein spots displayed quantitative changes in expression between normal and infarcted myocardial tissue. Eleven protein spots were subjected to mass spectrometry (MS) analysis and seven proteins were identified by peptide mass fingerprinting (PMF). These proteins may be involved in cardiovascular injury, and could play an important role in the treatment of coronary heart disease. __________ Translated from Chemical Journal of Chinese Universities, 2006, 8(27): 1467–1471 [译自: 高等学校化学学报]     

Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.     

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.     

大鼠骨髓间充质干细胞分化过程的比较蛋白质组学研究

从蛋白质组学角度分析大鼠骨髓间充质干细胞(MSCs)体外定向分化为心肌细胞过程中蛋白表达情况, 采用二维电泳分离蛋白, 用PDQuest软件分析蛋白表达差异, 并采用质谱(MALDI-TOF-MS)进行鉴定, 得到了54个蛋白点, 对蛋白的生物功能分析表明, 部分蛋白通过不同的信号途径参与了MSCs的分化过程.     

Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.     

Proteomic analysis of chondrocytes exposed to pressure

Chondrocytes are the only cell type present in mature articular cartilage (2-5% of total tissue). The biological activities of the chondrocyte population are regulated by genetic, biologic and biochemical factors, as well as environmental factors (stress, flow and electric field). Although compressive forces within joint articular cartilage are required for maintenance of the normal composition of articular cartilage, there is a lack of knowledge about the number of pressure-related proteins expressed in articular cartilage. Two-dimensional gel electrophoresis (2-DE) and high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC/ESI-MS/MS) were used to identify the levels of pressure-related proteins expressed by chondrocytes grown in the presence or absence of hydrostatic pressure. A total of 266 spots were excised from the gels and analyzed by HPLC/ESI-MS/MS. Functional classification of up-regulated proteins indicated that energy and protein fate were the main biological processes occurring in pressurized chondrocytes. Furthermore, membrane-bound transferrin-like protein p97, a marker of chondrocyte differentiation, was only expressed in chondrocytes under hydrostatic pressure. These data suggest that hydrostatic pressure can induce cell differentiation by increasing the expression level of energy metabolism- and protein fate-related proteins, indicating that hydrostatic pressure may be needed for normal biosynthesis and differentiation of articular chondrocytes.     

Proteome study of colorectal carcinogenesis

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.     

Systematic annotation and bioinformatics analyses of large-scale Oryza sativa proteome

Much has been now recognized on the rice (Oryza sativa L.) proteomics by using the powerful experimental tool two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE can be utilized for monitoring global changes of quantitative protein expression in specific tissues under various conditions. However, systematic annotations of the protein spots generated by 2D-PAGE are still limited for rice. In this study, a new approach for Oryza sativa proteome annotation based on the 2D-gel maps was developed. Based on the publicly available 2D-PAGE data of rice, 11,201 gel spots were annotated accounting for 87.2% of the total spots on the gel maps. Gel spot alignments were performed for the annotated gel maps belonging to 23 rice tissues or organelles. In summary, 253 alignments between 23 tissues or organelles were performed, and 26,207 co-expressed proteins were identified using our analytical strategy. Large-scale bi-cluster analysis of 23 tissues/organelles proteomes of rice was carried out to detect novel functional proteins. Function and pathway analysis identified a number of common gene products with great potential in regulating specific physiological and biochemical events within various rice tissues/organelles. It also suggested that the tissue- or organelle-specific proteins might be responsible for the functional divergence of these tissues or organelles. Taken together, this study provides us new strategies and informative resources for rice proteome research based on 2D-PAGE data.