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.     

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.     

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.     

应用蛋白质组学技术初步分析HeLa细胞饥饿诱导的自噬相关蛋白

应用双向凝胶电泳(2-DE)结合质谱技术研究饥饿诱导自噬的HeLa细胞(实验组)与正常培养的HeLa细胞(对照组)的差异表达蛋白质。结果显示对照组样本的2-DE图谱检测到1253±100个蛋白斑点,实验组样本检测到1216±125个蛋白斑点,二者主要斑点的位置与数量基本一致。对差异表达蛋白进行质谱分析,首次发现前折叠素2,转胶蛋白2,乳腺癌扩增序列2和Annexin A3在自噬细胞中表达上调,提示这4种蛋白可能参与饥饿诱导的细胞自噬。本研究为自噬的机制初步提供了线索。     

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.     

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.     

K-Means聚类分析法筛选柠檬香茅茎叶差异蛋白及鉴定

柠檬香茅含有大量的香茅精油,运用十分广泛,然而其茎、叶的精油含量却相差悬殊。 为探索柠檬香茅精油代谢相关的蛋白途径,本文对柠檬香茅旗叶、成熟叶及茎秆等材料进行精油含量、总蛋白含量测定及双向凝胶电泳(2-DE)表达谱分析,运用k-means聚类分析方法对2-DE电泳中差异蛋白斑点的丰度、等电点和相对分子质量进行聚类分析和讨论,结果表明,旗叶和茎秆上调表达的蛋白质斑点的聚类对于相对分子质量变化敏感,成熟叶上调表达蛋白质斑点对于丰度的变化较为敏感。 预测了精油代谢功能相关的蛋白质斑点15个,挖取预测蛋白质斑点通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/TOF-MS)成功鉴定了9个蛋白质。 本研究为柠檬香茅精油的蛋白代谢途径提供新的基础信息及研究思路。     

Human liver protein map: a reference database established by microsequencing and gel comparison.

This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.     

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.     

Composition and genetic variability of proteins from nuclear fractions of mouse (DBA/2J and C57BL/6J) liver and brain

Proteins from nuclear plasma of mouse liver and brain and from the nuclear membranes of mouse liver were separated by two-dimensional electrophoresis. For the purpose of comparison, liver cytosol proteins were also investigated. The protein samples were prepared from two inbred strains of the mouse (DBA/2J, C57BL/6J) and their hybrids. The patterns obtained were compared with regard to the composition and genetic variability (qualitative and quantitative variants) of proteins from different nuclear fractions and organs. The percentage (greater than 30%) of spots common to different organs (liver, brain), but from the same nuclear fraction (plasma) was greater than the percentage (less than 20%) of spots common to different cell and nuclear fractions (cytosol, nuclear plasma and nuclear membranes) of the same organ (liver). Quantitative genetic variants occurred much more frequently than qualitative genetic variants (5.1% vs. 0.2%; liver nuclear plasma). The incidence of genetic variants was much higher in liver (5.3%) than in brains (0.0%), and higher in solubilized nuclear proteins (5.3%) than in structure-bound nuclear proteins (2.1%).     

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.     

Identification of selenium-containing proteins in selenium-rich yeast aqueous extract by 2D gel electrophoresis, nanoHPLC-ICP MS and nanoHPLC-ESI MS/MS

An approach based on the consecutive use of nanoHPLC-ICP collision cell MS and nanoHPLC-electrospray MS was proposed for the analysis of water-soluble selenium-containing proteins in selenium-rich yeast after their separation by 2D gel electrophoresis (GE). An ultrasonic probe was employed for fast protein extraction avoiding sample heating and thus reducing the risk of protein degradation. The efficiency of different extraction steps were critically evaluated by total selenium analysis and size-exclusion chromatography (SEC)-ICP MS. Prior to electrophoresis proteins were purified by acetone precipitation. The protein-containing spots from 2D GE were excised and digested with trypsin. The digests obtained were analyzed by nanoHPLC-ICP MS in order to check for the presence of selenium-containing peptides; this allowed the detection of target proteins for further analyses (two out of five spots). The subsequent analyses of the selected digests by nanoHPLC-ES MS/MS allowed the attribution of amino acid sequences to peaks detected by ICP MS revealing the presence of two selenium-containing proteins: SIP 18 and HSP 12.     

Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry

In the previous study, the proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 were separated by high resolution two-dimensional electrophoresis (2-DE). Image analysis revealed that 99 protein spots showed quantitative and qualitative variations that were significant (P < 0.01) and reproducible. Here we report the identification results of some of these protein spots. Protein spots excised from 2-D gels were subjected to in-gel digestion with trypsin, and the resulting peptides were measured by microbore high performance liquid chromatography - ion trap - mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. Twelve protein spots were identified with high confidence using SEQUEST with uninterpreted MS/MS raw data. Besides inosine-5'-monophosphate dehydrogenase 2, heat shock 27 kDa protein, calreticulin and calmodulin, whose expression was elevated in hepatoma cells, glutathione-S-transferase P was identified from hepatoma cells in which its level was 18-fold higher compared to human liver cells. Two spots were identified as the homologs of reticulocalbin for the first time in hepatoma cells and their expression increased compared to liver cells. However, tubulin beta-1 chain and natural killer cell enhancing factor B were downregulated in hepatoma cells. A tumor suppressing serpin, maspin precursor, was identified from one spot whose quantity was much higher in the normal liver cell line. More interestingly, epidermal fatty acid-binding protein (E-FABP) and fatty acid-binding protein, adipocyte-type (A-FABP), were detected in liver cells but not in hepatoma cells. The functional implication of the identified proteins was discussed.     

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.     

Proteomics Dissection of Cold Responsive Proteins Based on PEG Fractionation in Arabidopsis

Proteome profiling was performed on Arabidopsis plant exposed to cold stress at 4 ℃ for 24 h in an attempt to explore the mechanisms of plant climate adaptation.The polyethylene glycol（PEG） fractionation protocol developed in this lab was used to identify as many differentially expressed low-abundance proteins as possible.In comparison with those of the biological controls,67 protein spots with at least two-fold difference in expression were identified for the plant exposed to cold temperatures; and from these spots,50 proteins were successfully identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry（MALDI-TOF MS）.Bioinformatics studies on these proteins show that 57.8％ of these proteins were localized in the chloroplast.Of these proteins,8 ones have functions in photosynthesis,including glycine hydroxymethyltransferase,Rubisco large subunit,Rubisco activase,PSBO2,fructose-1,6-bisphosphate aldolase,NADP-dependent malate dehydrogenase,sedoheptulose bisphosphatase and photosystem Ⅱ reaction center PsbP family protein,suggesting that photosynthesis is greatly affected by cold stress.The identified proteins were validated by quantitative real-time polymerase chain reaction（qPCR）.Taken together,our results suggest that the chloroplast might also act as a cold stress sensor and that photosynthesis-related proteins may play important roles in cold acclimation for Arabidopsis.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.