樟子松突变丛生枝蛋白质的双向电泳分析

目的：研究樟子松突变丛生枝的发生机理。方法：对其正常枝和突变丛生枝进行蛋白质双向电泳,通过ImageMaster2-DE计算机分析系统对结果进行定性、定量分析。结果：共有59个蛋白点在表达的质和量上有变化,其中35个蛋白点在突变丛生枝2-DE图谱中表达量均高于正常枝的蛋白点,20个蛋白点在突变丛生枝2-DE图谱中表达量均低于正常枝的蛋白点,4个蛋白点在突变丛生枝图谱上发生缺失。结论：差异表达的蛋白点可能与突变丛生枝的发生有关。     

家蚕蛹期雌雄生殖腺蛋白质双向电泳比较分析

分析家蚕Bombyx mori雌雄生殖腺细胞蛋白质,有利于发现性别分化相关的功能蛋白质,探讨生殖腺发育相关基因的表达调控机理。本研究利用蛋白质双向凝胶电泳和图像分析技术,分析家蚕蛹期第2天的雌雄生殖腺细胞蛋白质。结果表明: 在雄蚕生殖腺蛋白质电泳图谱中共检测到435个蛋白斑点,其中特异性蛋白斑点73个,占总蛋白斑点数的16.8％;雌蚕生殖腺的电泳图谱中有417个蛋白斑点,其中特异性蛋白斑点55个,占总蛋白斑点数的13.2％。雌雄能匹配的蛋白斑点有362对,匹配率达85.0％。     

HUPO BPP pilot study: a proteomics analysis of the mouse brain of different developmental stages

This study is a part of the HUPO Brain Proteome Project (BPP) pilot study, which aims at obtaining a reliable database of mouse brain proteome, at the comparison of techniques, laboratories, and approaches as well as at preparing subsequent proteome studies of neurologic diseases. The C57/Bl6 mouse brains of three developmental stages at embryonic day 16 (E16), postnatal day 7 (P7), and 8 wk (P56) (n = 5 in each group) were provided by the HUPO BPP executive committee. The whole brain proteins of each animal were individually prepared using 2-DE coupled with PDQuest software analysis. The protein spots representing developmentally related or stably expressed proteins were then prepared with in-gel digestion followed with MALDI-TOF/TOF MS/MS and analyzed using the MASCOT search engines to search the Swiss-Prot or NCBInr database. The 2-DE gel maps of the mouse brains of all of the developmental stages were obtained and submitted to the Data Collection Centre (DCC). The proteins alpha-enolase, stathmin, actin, C14orf166 homolog, 28,000 kDa heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40 S ribosomal protein S3a were successfully identified. A further Western blotting analysis demonstrated that enolase is a protein up-regulated in the mouse brain from embryonic stage to adult stage. These data are helpful for understanding the proteome changes in the development of the mouse brain.     

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.     

Comparative proteomic analysis of longan (Dimocarpus longan Lour.) seed abortion

Two-dimensional gel electrophoresis (2-DE), coupled with mass spectroscopy, was used to study seed abortion in Dimocarpus longan Lour. (cv. Minjiao 64-1) by comparing normal and aborted seeds at three developmental stages. More than 1,000 protein spots were reproducibly detected in 2-DE gels, with 43 protein spots being significantly altered in their intensity between normal and aborted seeds at least at one stage. Thirty-five proteins were identified by matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF-MS/MS) analysis and protein database searching. Most of the identified proteins were associated with a variety of functions, including energy and metabolism (30%), programed cell death (9%), antioxidative processes (14%), chaperonin (23%), cell division, amino acid metabolism, secondary metabolism, and other functional classes. Furthermore, the expression patterns of HSP70 and cytosolic ascorbate peroxidase (cAPX) were validated by immunoblotting analysis. This study provides a novel, global insight into proteomic differences between normal and aborted seeds in longan. We anticipate that identification of the differentially expressed proteins may lead to a better understanding of the molecular basis for seed abortion in longan.     

椰扁甲啮小蜂寄生后椰心叶甲蛹血淋巴蛋白质双向电泳图谱分析

分析昆虫被寄生蜂寄生后血淋巴蛋白质组分及组分含量变化,从蛋白质水平分析寄生蜂寄生引起寄主昆虫的生理变化,有利于探索寄生蜂调控寄主的机理。以椰心叶甲Brontispa longissima和椰扁甲啮小蜂Tetrastichus brontispae为研究材料,采用蛋白质双向电泳技术及图像分析技术分析了椰心叶甲蛹被椰扁甲啮小蜂寄生后不同时期血淋巴蛋白质图谱及其变化情况。结果表明:寄生后0.5, 1, 2, 3和4 d,被寄生蛹血淋巴共检测到的蛋白斑点数分别为982, 926, 712, 636和680个,其中特异性蛋白斑点的数量分别为650, 400, 336, 229和150个;同期未被寄生蛹血淋巴共检测到的到的蛋白斑点数分别为645, 817, 640, 873和940个,其中特异性蛋白斑点的数量分别为313, 291, 264, 466和410个;被寄生蛹和同期未被寄生蛹能匹配的蛋白斑点分别为332, 526, 376, 407和530对。被寄生蛹血淋巴蛋白斑点数量的异常变化表明寄主的生理功能受到了严重的影响。     

溴氰菊酯胁迫下小菜蛾幼虫体内差异表达蛋白的分析

小菜蛾Plutella xylostella L.是世界性十字花科蔬菜的主要害虫, 已对多种杀虫剂产生抗性, 其中以对拟除虫菊酯类杀虫剂的抗性发展最快。溴氰菊酯是拟除虫菊酯杀虫剂中杀虫毒力最强的品种。我们前期的研究发现, 小菜蛾溴氰菊酯敏感品系（DS）和抗性品系（DR）成虫期的蛋白质双向电泳(2-DE)图谱存在显著差异。本研究通过双向电泳技术从小菜蛾4龄幼虫中分离出89个有明显差异的蛋白点, 从中选出30个进行串联质谱(MALDI-TOF-MS)实验, 并利用蛋白质数据库检索这些在抗性品系中表达而在敏感品系中不表达或者不同品系中差异表达的蛋白质的归属、 性质和功能, 最终成功鉴定出10个蛋白。对其中的3个基因进行了荧光定量PCR验证, 发现这些蛋白质在mRNA水平的表达与在蛋白水平的表达是一致的。这些在溴氰菊酯胁迫下差异表达的蛋白为研究溴氰菊酯的作用靶标和作用机理, 以及筛选与其抗性相关的蛋白质提供了依据。     

Geometric algorithms for the analysis of 2D-electrophoresis gels.

In proteomics, two-dimensional gel electrophoresis (2-DE) is a separation technique for proteins. The resulting protein spots can be identified either by using picking robots and subsequent mass spectrometry or by visual cross inspection of a new gel image with an already analyzed master gel. Difficulties especially arise from inherent noise and irregular geometric distortions in 2-DE images. Aiming at the automated analysis of large series of 2-DE images, or at the even more difficult interlaboratory gel comparisons, the bottleneck is to solve the two most basic algorithmic problems with high quality: Identifying protein spots and computing a matching between two images. For the development of the analysis software CAROl at Freie Universit?t Berlin, we have reconsidered these two problems and obtained new solutions which rely on methods from computational geometry. Their novelties are: 1. Spot detection is also possible for complex regions formed by several "merged" (usually saturated) spots; 2. User-defined landmarks are not necessary for the matching. Furthermore, images for comparison are allowed to represent different parts of the entire protein pattern, which only partially "overlap." The implementation is done in a client server architecture to allow queries via the internet. We also discuss and point at related theoretical questions in computational geometry.     

不同上样方式对蛋白质组双向电泳图谱质量的影响比较

蛋白质组技术难点之一是如何获取尽可能多的细胞或组织的蛋白信息.双向电泳蛋白斑点数目直接反映了实验蛋白质组信息的完整性.除样品制备外,蛋白上样方式对双向电泳图谱的质量和完整性有直接的影响.实验论文从以下3个方面考察不同的蛋白上样方式对双向电泳图谱的影响;即：水化上样与杯上样;一次上样与重复上样;以及酸性端加样与碱性端上样.实验结果发现;蛋白上样量较大时,杯上样方式的图谱斑点数目较水化上样方式明显增多;样品蛋白浓度较高时,稀释多次上样明显优于一次性浓缩蛋白上样;蛋白裂解液(MCF7乳腺癌细胞)在酸性端加样对偏碱性蛋白的分离未发现明显优势.相反,在等电聚焦伏小时(Vh) 足够的前提下,碱性端加样对偏碱性端蛋白反而有利,表现为斑点数目较多,而且等电点方向拖尾减轻.实验结果对提高双向电泳的质量以及相关蛋白质组信息的完整性提供了有益的技术参考.     

Variation in the holm oak leaf proteome at different plant developmental stages, between provenances and in response to drought stress

Major proteins of the holm oak leaf proteome have been previously identified using a combination of 2-DE, MS analysis and BLAST similarity search (Jorge et al., Proteomics 2005, 5, 222-234). That study, conducted with field samples from mature trees, revealed the existence of a great variability in the 2-DE protein map, with qualitative as well as quantitative changes, both analytical and biological. A similar study has been carried out with 2-year-old seedlings to analyze and study: (i) changes in the 2-DE protein profile at different tree developmental stages; (ii) the 2-DE protein map variability between three different Spanish provenances; and (iii) variations in the 2-DE protein profile in response to drought stress. Although the protein profile of leaves from seedlings and mature trees was fairly similar, the biological variance found was lower in the former. In the present study, new proteins have been identified. At least four different protein spots differentiated Spanish provenances, two of them identified as an ATP synthase alpha chain, and a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase. Fourteen different protein spots were qualitatively variable between well-watered and drought-stressed seedlings, with some of them corresponding to enzymes of carbohydrate and protein metabolism. Data presented indicated the mobilization of storage proteins and carbohydrates, as well as photosynthesis inhibition under drought conditions.     

Evaluation of desorption of proteins adsorbed to hydrophilic surfaces by two-dimensional electrophoresis

The evaluation of the plasma protein adsorption patterns of superparamagnetic iron oxide (SPIO) particles is of high interest concerning their in vivo fate and is carried out by two-dimensional electrophoresis (2-DE). The sample preparation is of great importance, especially the removal of the adsorbed proteins (desorption) from the particle surface for subsequent analysis by 2-DE. The removal is carried out by a desorption solution. In this study, negatively and positively charged SPIO model particles were under investigation concerning the desorption of proteins adsorbed on their surfaces. Firstly, the desorption process was determined quantitatively using the Bradford protein assay. Secondly, the removable or nonremovable protein species, from particles surface were under investigation by 2-DE. Looking at the desorption in a quantitative manner with the Bradford assay, the desorption efficacy from negatively charged particles was about 90%. In the case of the positively charged particles, the desorption efficacy seemed to be reduced, approximately 34% of the proteins remained on the surface. Comparing the protein patterns of the particles evaluated by 2-DE in the desorption solution and the proteins remaining on the particles, they confirmed the results from the protein quantification. After desorption, the IgG gamma-chains were found to be the dominant protein fraction remaining on the negatively charged particles. On the positively charged particles, many more protein species were found after desorption. The more basic the protein fragments, the more ineffective was the desorption from the positively charged model particle, and vice versa. Nevertheless, all protein spots were found qualitatively in the desorption solution, especially when the desorption solutions still containing the particles were used for the 2-DE analysis. In conclusion, 2-DE could be confirmed as the "gold standard" for determining the plasma protein adsorption patterns of nanoparticulate systems.     

橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。     

Proteomic Screening of Antigenic Proteins from the Hard Tick,Haemaphysalis longicornis (Acari: Ixodidae)

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

A proteomic approach to identify developmentally regulated proteins in Leishmania infantum

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.     

糠秕马拉色菌酵母态和菌丝态蛋白差异表达的研究

目的通过双向电泳及串联质谱技术鉴定糠秕马拉色菌酵母态及菌丝态差异蛋白,在蛋白水平探讨两态转化机制及致病机理。方法分别诱导糠秕马拉色菌标准株酵母态和菌丝态菌体,利用玻璃珠研磨和超声波破碎细胞壁,三氯乙酸/丙酮沉淀获取总蛋白。双向电泳分离蛋白,PDQuest软件比对找出差异蛋白点。电喷雾串联质谱对差异点进行肽段测序,用Mascot和NCBI的Blast软件经蛋白质数据库鉴定蛋白质。结果经双向电泳分离的糠秕马拉色菌酵母态、菌丝态蛋白各有800多个蛋白点、64个蛋白点表达量有3倍以上差异,其中11个为酵母态特有,9个菌丝态特有。在选取的40个差异点中,成功鉴定出22个点,共16个蛋白。经Mascot和Blast软件检索,有明确功能的蛋白中,肌动蛋白、丝切蛋白等9个蛋白在菌丝态上调,谷胱甘肽转移酶、细胞支架信号蛋白等5个蛋白下调。结论鉴定出16个蛋白分别与细胞代谢、运动、氧化应激等功能相关,为了解糠秕马拉色菌表型转换机制和致病机理提供重要信息。     

Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells

Regulated proteolysis plays important roles in cell biology and pathological conditions. A crosstalk exists between apoptosis and the ubiquitin?Cproteasome system, two pathways responsible for regulated proteolysis executed by different proteases. To investigate whether the apoptotic process also affects the 20S proteasome, we performed three independent SILAC-based quantitative proteome approaches: 1-DE/MALDI-MS, small 2-DE/MALDI-MS and large 2-DE/nano-LC?CESI?CMS. Taking the results of all experiments together, no quantitative changes were observed for the ??- and ??-subunits of the 20S proteasome except for subunit ??7. This protein was identified in two protein spots with a down-regulation of the more acidic protein species (??7a) and up-regulation of the more basic protein species (??7b) during apoptosis. The difference in these two ??7 protein species could be attributed to oxidation of cysteine-41 to cysteine sulfonic acid and phosphorylation at serine-250 near the C terminus in ??7a, whereas these modifications were missing in ??7b. These results pointed to the biological significance of posttranslational modifications of proteasome subunit ??7 after induction of apoptosis.     

多子房小麦蛋白质双向电泳体系的建立及优化

为建立适用于显性多子房小麦细胞质效应的蛋白质双向电泳体系,以显性多子房小麦材料DUOII与特异细胞质材料TeZhiI杂交的F1幼穗为材料,采用TCA-丙酮法提取蛋白质,并在IPG胶条长度和pH范围、SDS-PAGE凝胶浓度及蛋白质上样量等方面,对多子房小麦幼穗蛋白质双向电泳体系进行了探究与优化.结果表明,本文采用的蛋白质定量方法准确度高(R²=0.9999),确立了17 cm, pH4～7的IPG胶条, 12% SDS-PAGE分离胶,上样量为900 μg的双向电泳方法体系,获得了最适合本研究蛋白质组分析的双向电泳图谱. 经PDQuest 2DE 8.0.1软件分析,2-DE图谱上可分辨出1.444±14个清晰蛋白质点,且重复性较高(95%), 相关系数为0.960. 建立了一套适用于显性多子房小麦细胞质效应研究的蛋白质双向电泳体系.