差速离心结合蛋白质组学技术研究受镉盐胁迫后的牙鲆肝差异蛋白质

人工构建镉盐污染源,选用差速离心结合双向凝胶电泳(2D-PAGE)法,高效提取、分离和筛选牙鲆(Paralichthys olivaceus, PO)受镉盐胁迫后的肝脏全蛋白和差异蛋白质.实验结果表明: 选用直接裂解法提取牙鲆肝(PO liver POL)全蛋白质且用2D-PAGE分离,可获得约800个蛋白斑点,其中镉盐诱导了11个差异蛋白斑点.以相对离心力为1000×g、12000×g和100000 ×g 的差速离心法,分别制备了3种沉淀蛋白和1种胞浆蛋白,称为POL组分Ⅰ、POL组分Ⅱ、POL组分Ⅲ和POL组分Ⅳ(胞浆蛋白),蛋白斑点数目分别为380、550、500和850个,总计2280个,明显高于直接裂解法.比较分析法发现,差速离心结合2D-PAGE分离技术可获得牙鲆肝脏受镉盐胁迫后表达的54个差异蛋白质,并适合于用肽质量指纹(peptide mass fingerprint,PMF)图谱技术鉴定.本实验所建立的差速离心结合蛋白质组学技术可高效提取、分离和鉴定组织全蛋白或差异蛋白,并能有效地筛选出蛋白指示物.     

在镉盐胁迫下扇贝鳃组织应激蛋白的研究

采用透射电子显微镜观察了虾夷盘扇贝(Patinopecten yessoensis)鳃组织细胞的超微结构, 发现镉盐能胁迫鳃组织中的腮丝、细胞核和线粒体产生病变. 利用双向凝胶电泳(2D-PAGE)优化分离扇贝鳃组织的全蛋白, 获得约800个蛋白质斑点, 并筛选出37个由于镉盐胁迫而产生的差异蛋白质斑点. 选用基质辅助激光解吸离子化-飞行时间质谱(MALDI-TOF MS)技术和数据库检索鉴定差异蛋白, 结果发现7个与镉毒性密切相关的蛋白质, 即热休克蛋白70和β-淀粉酶等上调蛋白质及原肌球蛋白、肌动蛋白和钙活化核苷酸酶1等下调蛋白质. 此外, 还发现转录调节子Crp/Fnr家族为低表达蛋白质, 而ABC转运子为高表达蛋白质. 在这些差异蛋白中, 部分蛋白质适合作为连续监测流动海水中镉污染程度及评价其危害性的蛋白指示物.     

蛋白质组学技术筛选与鉴定鲤鱼嗅脑急性创伤后的应激蛋白质

优化建立鲤鱼(cyprinus carpio,CC)嗅觉端脑(嗅脑)全蛋白提取技术。联用低渗透裂解和液氮冻溶法破碎鲤鱼嗅脑组织(rhinencephalon tissue of cyprinus carpio,CCRT)、低速离心提取CCRT全蛋白,并采用双向凝胶电泳(2D-PAGE)技术进行有效分离。经分析与统计,每张CCRT的2D-PAGE图谱中的蛋白质斑点数目约为1200个。分别分离CCRT的脂溶性和水溶性全蛋白,并获得高分辨率的2D-PAGE图谱。选用差异蛋白质组学技术筛选经10%冰醋酸创伤后的CC,其端脑组织所表达出的6种应激蛋白质,并用肽质量指纹谱(peptide mass fingerprinting,PMF)和数据库检索技术给予鉴定。其中3种蛋白质为70S热休克蛋白、β微管蛋白和DNA链接酶IV,有望作为研究大脑急性创伤后的应激修复途径和机理的指示蛋白质。     

在镉盐胁迫下用蛋白质组学技术筛选与鉴定海兔亚口腔神经节的差异蛋白质

以蓝斑背肛海兔(Notarcus leachii cirrosus Stimpson, NLCS)的口腔神经节(Buccal ganglion, BG)为研究对象, 按BG形态对称性, 解剖成亚BG(sub-BG, SBG), 并分为左SBG和右SBG, 简称为LSBG和RSBG. 用双向凝胶电泳(2D-PAGE)技术优化分离LSBG和RSBG全蛋白质, 并采用蛋白质组学和数据库比对技术筛选与鉴定差异蛋白质. 实验结果表明, LSBG和RSBG之间的差异蛋白质主要由活性多肽的前体蛋白或降解后大片段多肽组成, 它们对维持BG的生理功能起着重要的作用. 在急性镉盐(10 μg/mL)胁迫下, NLCS的LSBG和RSBG表达了由镉盐诱导的差异蛋白质, 并采用蛋白质组学技术分别分离、筛选和鉴定, 其主要的差异蛋白质有下调的肌球蛋白、钙结合蛋白、上调的热休克蛋白和硫氧还蛋白. 这些蛋白质可能与BG细胞抗镉毒性有关, 部分差异蛋白质适合于监测镉盐污染且开展毒理学研究的蛋白指示物.     

温度对线虫的砷胁迫应答的影响

应用免疫印迹法(Western blots)与酶联免疫吸附法(ELISA)检测了不同强度砷胁迫和热影响下秀丽隐杆线虫热休克蛋白Hsp90表达。免疫印迹法通过PVDF膜对蛋白带显色,酶联免疫吸附法使用酶标仪测定蛋白光密度值;蛋白表达水平以相对值表示。冷冲击增强了线虫对砷胁迫的抗性,而高温显著降低了线虫对As的耐受性;砷胁迫下线虫Hsp90为分子量为90 kDa的应激蛋白,其具有应激响应。免疫印迹法和酶联免疫法对不同浓度砷胁迫下线虫Hsp90表达的分析结果存在差异,应用酶联免疫法检测中低等强度胁迫下Hsp90表达具有较高灵敏度。     

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.     

Ⅱ型糖尿病大鼠神经视网膜组织中差异蛋白质分析

以高脂饮食联合小剂量链脲菌素(Streptozotocin, STZ ) 注射方法诱导大鼠II型糖尿病 (T2DM) 模型为研究对象, 采用比较蛋白质组学技术鉴定和分析早期糖尿病神经视网膜差异表达蛋白.通过联用液氮内研磨、冰浴超声、高速离心提取全蛋白技术,提取每只大鼠神经视网膜全蛋白质总量约900 μg.采用双向凝胶电泳(2-DE) 分离技术,获得2500个以上可辩认的蛋白质斑点.通过蛋白质组学比对技术筛选出糖尿病状态下神经视网膜差异表达蛋白,用基质辅助激光解吸电离飞行时间串联质谱 (MALDI-TOF- MS/MS) 和肽质量指纹图谱 (PMF) 鉴定出20个差异蛋白点.按照Gene Ontology (GO)分类体系,对差异蛋白归类分析,揭示其亚细胞定位和分子功能.应用Pathway Studio软件对差异表达蛋白的生物学意义进行分析,认为凋亡是早期糖尿病视网膜病变(Diabetic retinopathy,DR) 重要的细胞事件;AnnexinΙ、CRYAB、mtHsp70蛋白是与病理过程相关的关键蛋白,对研究DR致病机制有重要意义.     

双向电泳-串联飞行时间质谱对急性脊髓损伤大鼠差异蛋白质的分离鉴定

为研究急性脊髓损伤的病理和修复机制,建立了正常和急性脊髓损伤大鼠脊髓组织的双向电泳图谱,确认了16个变化3倍以上的差异蛋白斑点,其中在急性脊髓损伤组蛋白斑点表达上调的10个,表达下调的6个.利用基质辅助激光解析电离串联飞行时间质谱(MALDI-TOF/TOF-MS)成功鉴定出16个差异蛋白质,其中,6个蛋白质是新鉴定的,2个蛋白为同一蛋白的不同修饰.除髓磷脂碱性蛋白(MBP)和中相对分子质量神经丝蛋白(NF-M)已证实与脊髓损伤密切相关外,其它蛋白与脊髓损伤的关系有待研究.     

鼠肝癌淋巴道转移细胞模型的蛋白质组学研究

对2株来源于同一亲本细胞但淋巴道转移力显著不同的小鼠肝癌腹水型细胞株Hca-F(淋巴结转移率75%)和Hca-P(淋巴结转移率25%), 采用荧光差异双向凝胶电泳(2D DIGE)和DeCyder定量分析软件及HPLC-nESI-MS/MS技术, 定量分析和鉴定了小鼠肝癌细胞Hca-F和Hca-P的差异表达蛋白. 结果显示, 有116个蛋白质点表达水平存在明显差异(p<0.05), 在Hca-F中表达上调蛋白质点62个, 下调蛋白质点54个. 对所有116个蛋白质点进行了电喷雾串联质谱鉴定, 共鉴定出109种单一(Unique)蛋白. 其中部分蛋白已被报道与不同类型肿瘤的发生、浸润和转移相关, 多数蛋白质被首次报道与肝癌的淋巴道转移过程直接相关.     

拟南芥和甜菜夜蛾相互作用的差异蛋白分析

以甜菜夜蛾(Spodoptera exigua)和模式植物拟南芥(Arabidopsis thanliana)作为研究体系, 应用蛋白质双向凝胶电泳(Two-dimensional gel electrophoresis, 2-DE)分析了在甜菜夜蛾取食诱导条件下拟南芥蛋白表达的差异, 从蛋白质水平揭示昆虫取食诱导条件下植物的化学防御机制. 结果发现, 在昆虫取食诱导条件下, 有28个蛋白发生显著变化, 其中17个蛋白点上调表达, 11个蛋白点下调表达. 利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对差异蛋白进行了鉴定, 结果发现转酮酶、S-腺苷甲硫氨酸合成酶、二氢硫辛酰胺脱氢酶和脂肪酸合成酶在植物诱导化学防御中具有重要的作用, 其中脂肪酸合成酶与茉莉酸代谢通路相关.     

Differential Proteins of the Optic Ganglion in Octopus vulgaris Under Methanol Stress Revealed Using Proteomics

An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.     

蛋白质组学技术鉴定与分析三七粉诱导海兔神经连索表达的差异蛋白质

RP-HPLC分离三七粉提取液,并鉴定含有Rb1、Rg1、Re、R1等皂甙成分。以蓝斑背肛海兔(Notarcusleachii cirrosus Stimpson,NLCS)为分析模型,三七粉提取液为诱导剂,选用蛋白质组技术研究NLCS神经连索诱导前后所表达的差异蛋白质。通过优化双向凝胶电泳分离NLCS神经连索全蛋白质组技术,获得496个蛋白质斑点。采用肽指纹图谱技术和数据库检索比对法,初步鉴定了NLCS受三七粉提取液诱导前后,其神经连索表达13个差异蛋白质,其中较高的匹配率蛋白质为肌动蛋白、3-羟酯酰辅酶A脱氢酶、ATP结合转运子和甲基转移酶12。选用LOC tree软件对13个差异蛋白质进行亚细胞定位,认为它们在保护神经系统中发挥重要的调节作用。     

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.     

Capillary column high-performance liquid chromatographic-electrospray ionization triple-stage quadrupole mass spectrometric analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis application to cerebellar protein mapping

A method is presented for the structural characterization of proteins separated by two-dimensional poly-acrylamide gel electrophoresis (2D-PAGE). The method includes separation of a protein mixture by 2D-PAGE, recovery of proteins from the gel spots revealed by copper staining and analysis of the proteins by triple-stage quadrupole mass spectrometry using an electrospray ionization interface (ESI-TSQMS). Prior to the mass spectrometric analysis, the extracted proteins were passed through a small reversed-phase column (10 × 4.0 mm I.D.) to remove salts and gel-derived contaminants and then introduced into the mass spectrometer through a reversed-phase capillary column with 0.25 mm I.D. Application of the method to the analysis of rat cerebellar proteins suggests that the molecular mass could be accurately determined with sub-picomole amounts of protein samples derived from one or two 2D gels. The method was also useful for peptide mapping and determination of amino acid sequences of proteins micro-prepared from the 2D gel. Because 2D-PAGE has an excellent resolving power in protein separation and because capillary LC-ESI-TSQMS provides structural information with very small amounts of samples, the combined system of 2D-PAGE and capillary LC-ESI-TSQMS described here should allow wide applications to molecular studies of genes and proteins, such as identifications of protein spots on 2D gels, confirmation of gene/protein sequences and analysis of post-translational modification of proteins present naturally in tissue/cell extracts or expressed by recombinant DNA techniques.     

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

优化分离与鉴定蓝斑背肛海兔口腔神经节蛋白质组

采用双向凝胶电泳技术优化分离蓝斑背肛海兔(Notarcus leachii cirrosus Stimpson, NLCS)口腔神经节(Buccal Ganglion, BG)蛋白质组, 并获得约300个蛋白质斑点. 用组合基质辅助激光解吸电离化飞行时间(MALDI-TOF)质谱技术和胶内酶解技术测定BG蛋白质组中的96个蛋白质斑点的肽指纹(Peptide mass fingerprint, PMF)图谱. 经数据库检索与比对后, 发现96种蛋白质中仅有4种蛋白质可获得较高的匹配率, 它们分别是微管蛋白(Tubulin)、 肌动蛋白(Actin)和两个1,5-二磷酸核酮糖-羧化酶/加氧酶(Ribulose-1,5-bisphosphate carboxylase/oxygenase, Ribulose), 均属于神经细胞骨架蛋白质; 同时还发现一种交配信号肽前体(Peptide mating pheromone precursor). 利用LOCtrees软件和分类法对56种蛋白质进行亚细胞定位与分类.     

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.