Two-dimensional capillary electrophoresis involving capillary isoelectric focusing and capillary zone electrophoresis

Capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) was on-line hyphenated by a dialysis interface to achieve a 2D capillary electrophoresis (CE) system. The system was used with just one high-voltage power supply and three electrodes (one cathode shared by the two dimensions). The focused zone in the first dimension (i.e. the cIEF) was driven to the dialysis interface by electroosmotic flow (EOF), besides chemical mobilization from the first anode to the shared cathode. And then in the second dimension (i.e. the CZE), the separated zone was further separated and driven by an inverted EOF, which originated from the charged layer of a cationic surfactant adsorbed onto the inner wall of the capillary. Finally, a solution of ribonuclease was rapidly separated to assess the feasibility of the two-dimensional CE implement. (C) 2003 Elsevier B.V. All rights reserved.

光敏核不育水稻叶片蛋白质的双向电泳分析及苗期的光周期反应

光敏核不育水稻农垦58S由晚粳农垦58突变形成。具有在适宜温度条件下，长日照诱导雄性不育、短日照诱导雄性可育的基本特性。光敏核不育水稻育性转换机理的阐明是两系法杂交稻技术的关键。 1.克隆光敏不育基因是研究光敏核不育水稻育性转换机理的一个重要方面，本文对通过反映农垦58S和农垦58遗传背景差异的蛋白质或受光周期调节的蛋白质实现克隆光敏不育基因的策略进行了可行性研究，得到以下结果： (1).利用双向电泳技术在光敏核不育水稻是58S叶片中发现一个不存在于农垦58的蛋白质，其分子量为59.8kDa，等电点pH为5.9（称为Pa），该蛋白的存在不受光照条件、发育时期的影响，反映出农垦58S与农垦58遗传背景的差异。 (2).Pa蛋白与农垦58S叶绿体P61蛋白具有相同的分子量、等电点和N－端氨基酸顺序，在不同品种水稻中具有相同的分布，因此它们很可能是同一个蛋白质分子。 (3).利用双向电泳技术发现P61(Pa)和P41蛋白不仅存在于光敏不育系中，也存在于常规可育粳稻中，与光敏不育性状没有平行关系。 (4).利用双向电脉技术发现10天14小时长日照能在农垦58S和农垦58中诱导一个分子量为36kDa、等电点pH为5.2的蛋白质（称为P_b)，该蛋白的表达受光敏色素的调控。因此P61（Pa）、P41及P_b蛋白均与光敏不育性状无直接关系，推测克隆这些蛋白的基因无法直接获得光敏不育基因。 2.在育性转换光周期敏感期已经发现长日照使农垦58S叶绿体发育不良，但在苗期光周期敏感期内，目前尚不知长日照是否会有同样的效应。本文以光周期对农垦58S苗期叶绿体发育的影响为主要内容，研究了农垦58S苗期的光周期反应，得到以下结果： (1).农垦58S从5叶龄期至6叶龄期开始对光周期敏感，短日照开始能诱导茎尖分化幼穗。 (2).不同的光周期对农垦58S 4叶龄期新展叶片叶绿体发育的影响无明显差异，叶结体结构与功能均表现正常。 (3).不同的光周期对农垦58S 6叶龄期新展叶片叶绿体发育的影响有明显差异。与短日照相比，长日照引起农垦58S部分叶绿体发育不良，导致光化学活性减弱、超分子结构异常。长日光周期对农垦58S叶绿体发育的不良效应可能是光周期敏感期内存在的一种特殊现象。

青杨组(Populus Section Tacamahaca Spach)不同种对非生物胁迫的响应差异

土壤是人类赖以生存的自然环境和农业生产的重要资源，目前土壤受到干旱和盐胁迫的危害越来越严重。杨树具有适应性强、生长快和丰产等特性，本论文以青杨组杨树为模式植物，研究杨树对土壤干旱和盐胁迫的生态生理及蛋白质组学反应，研究成果可为我国干旱半干旱地区营造人工林、防止沙漠化提供理论依据，也为恢复与重建盐污染地区退化生态系统提供科学指导。主要研究结果如下： 1 青杨不同种对逐步干旱胁迫的响应差异 将来自喜马拉雅山东缘高海拔的康定杨和低海拔的青杨枝条扦插在温室中，用来检测它们对逐步干旱胁迫的响应。研究结果表明来自不同海拔的杨树对逐步干旱胁迫的适应性反应是不一样的。株高、叶片发育、叶片相对含水量、丙二醛、过氧化氢等指标的显著性变化在青杨中比在康定杨中来得早些，而且随着干旱胁迫程度的增加，这些参数的变化越来越明显，尤其是当青杨受到严重干旱胁迫的时候；而可溶性蛋白、可溶性糖、游离脯氨酸、抗氧化酶活力变化在康定杨中来得早一些。与青杨相比，在干旱胁迫下，康定杨仍能保持较好的植株生长和叶片发育；康定杨也能在逐步干旱条件下积累更多的可溶性蛋白、可溶性糖、游离脯氨酸及抗氧化酶活力，但是在丙二醛和过氧化氢含量方面增加的更少些。而且，我们的研究结果表明高海拔的康定杨有更强的耐干旱能力，杨树对干旱胁迫的适应能力与干旱发生的速度、强度、持续时间及两种杨树的海拔有关。 2 干旱胁迫下青杨不同种的蛋白质组学分析 来自青杨和康定杨雌株的枝条扦插在温室中，用来研究它们对干旱胁迫的蛋白质组学反应。采用TCA-丙酮/酚提取法提取总蛋白，并进行双向电泳分析。在每个处理的重复图像中都能检测到1,000 个以上的蛋白点。在青杨中有58 个蛋白在干旱处理后发生显著变化，其中22 个蛋白通过肽指纹图谱成功鉴定。康定杨中有69 个蛋白的表达量发生了显著变化，其中有25 个蛋白通过肽指纹图谱成功鉴定。这些被鉴定的蛋白主要参与了光合作用、氧化还原平衡、信号传导、能量代谢、蛋白质合成等过程。尽管被鉴定的蛋白只占叶片总蛋白的很少一部分，但这些被鉴定的干旱响应蛋白可能对维持植株内部平衡方面有重要作用。 3 青杨的盐胁迫响应 青杨植株分别用 0、50 和100 mM NaCl 溶液进行处理。叶片相对含水量、叶绿素a、b 含量、CO2 同化速率和气孔导度的降低表明叶绿体受到了盐胁迫的影响。过氧化氢、丙二醛含量及电导率的升高表明细胞受到了伤害。可溶性糖、游离脯氨酸含量及抗氧化酶含量的上升增加了植株耐盐胁迫的能力。在每个处理的重复图像中都能检测到1,000 个以上的蛋白点。其中有38 个盐响应蛋白被成功鉴定，有16 个蛋白(点4、10、11、14、15、21、24、26、27、28、33、34、35、36、37 和38)出现在盐胁迫的植株中；3 个蛋白(点10、11 和35)只出现在重度盐胁迫处理中；而1 个蛋白(点1)只出现在对照处理中。2 个蛋白(点1 和2)表达量下降，其余蛋白点表达量都增加。被鉴定的蛋白一部分参与了生理生化反应，而另一部分则在信号传导、蛋白质合成等方面有重要作用。盐胁迫下的生理生化变化及蛋白质组学的联合研究有利于青杨对盐胁迫的适应性分析。 Soil is the indispensable environment for human survival and important resource for agriculture development. Nowadays soil is threatened by drought stress and salt stress. Poplars (Populus spp.) possess some characters such as strong acclimilation, fast growth and great production of biomass. In this study, different species of Populus section Tacamahaca spach were used as model plants to investigate the ecophysiological and proteomic responses to drought stress and salt stress. Our results can provide theoretical evidence for the afforestation and prevention of desertification in the arid and semi-arid areas, and also can supply scientific direction for the reconstruction and rehalibitation of ecosystems contaminated by salinity. The results are as follows: 1 Adaptive responses to progressive drought stress in two contrasting poplar species originating from different altitudes Cuttings of Populus kangdingensis C. Wang et Tung and Populus cathayana Rehd., originating from high and low altitudes in the eastern Himalaya, respectively, were examined during one growing season in a greenhouse to determine the effects of progressive drought stress. The results manifested that the adaptive responses to progressive drought stress were different in these two species from different altitudes. Significant changes in height increment, leaf development, relative water content (RWC), malondialdehyde (MDA) and hydrogen peroxide (H2O2) appeared earlier in P. cathayana than in P. kangdingensis, whereas changes in soluble protein, soluble sugar, free proline and antioxidant enzymes appeared earlier in P. kangdingensis. In addition, changes in these parameters became more and more significant when the drought stress progressed, especially under severe drought stress in P. cathayana. Compared with P. cathayana, P. kangdingensis was able to maintain a superior height increase and leaf development under drought stress. Also, P. kangdingensis possessed greater increments in soluble protein, soluble sugar, free proline and antioxidant enzymes, but lower increments in MDA and H2O2 than did P. cathayana when the cuttings were exposed to progressive drought stress. Our results suggest that P. kangdingensis originating from the high altitude has a better drought tolerance than does P. cathayana originating from the low altitude. Furthermore, this study manifested that acclimation to drought stress are related the rapidity, severity, duration of the drought event and the altitude of two contrasting species. 2 Proteomic responses to drought stress in two contrasting poplar species originating from different altitudes The cuttings from a female clone of P. kangdingensis and P. cathayana were used to determine proteomic response to drought stress, respectively. Total proteins of the leaves were extracted by a combination of TCA-acetone and phenol, and separated by two-dimensional gel electrophoresis. More than 1,000 protein spots were reproducibly detected on each gel. 58 differentially expressed spots were detected under drought stress in P. cathayana and 22 drought-responsive proteins were identified by peptide mass fingerprint. 69 differentially expressed spots were detected under drought stress in P. kangdingensiss and 25 drought-responsive proteins were identified by peptide mass fingerprint. The identified proteins are involved in several processes, i.e., signal transduction, protein processing, redox homeostasis, CO2 fixation and energy metabolism. Although the proteins identified in this investigation represent only a very small part of the poplar leaf proteins, some of the novel drought-responsive proteins identified here may be involved in the establishment of homeostasis in response to drought stress in the woody plants. 3 Responses to salt stress in P. cathayana Cuttings from a female clone of P. cathayana were treated by Hoagland’s solution: 0, 50, 100 mM NaCl, respectively. Salinity significantly decreased the relative water content of leaves, the contents of chlorophyll a and chlorophyll b, CO2 assimilation rate (A) and stomatal conductance (gs) in both salt stress treatments，which suggested the chloroplast was affected by salt stress. The observed increases of H2O2 and malondialdehyde contents and electrolyte leakage suggested that salinity caused cellular damage, whereas the increases in compatible solutes and in the activities of antioxidant enzymes enhanced the salt tolerance. More than 1,000 protein spots were reproducibly detected on each gel, and 38 salt-responsive proteins were successfully identified by peptide mass fingerprint (PMF). 16 spots (spot 4, 10, 11, 14, 15, 21, 24, 26, 27, 28, 33, 34, 35, 36, 37 and 38) absent in the control sample were induced by the salt treatment, and three spots (spot 10，11 and 35) were present only in the severely salt-stressed treatment. The %vol of the differentially expressed proteins generally increased with progressing salt stress, except for the decreased %vol of two proteins (spot 1 and 2) under salt stress and the presence of spot 1 only in the control sample. Some of the novel salt-responsive proteins identified here may be involved in physiological, biochemical response to salt stress in P. cathayana, the other identified proteins play a role in numerous cellular functions, including signal transduction and protein processing. An integrated physiological, biochemical and proteomic approach was used here to systematically investigate salt acclimation in poplar.

High resolution mass spectrometric alveolar proteomics: Identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micro preparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products.

The encysted dormant embryo proteome of Artemia sinica

The possibility of the brine shrimp Artemia to produce dormant embryo (cysts) in diapause is a key feature in its life history. In the present study, we obtained a proteomic reference map for the diapause embryo of Artemia sinica using two-dimensional gel electrophoresis with a pH range of 4-7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected, and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these, 39 spots representing 33 unique proteins were identified, which are categorized into functional groups, including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. This reference map will contribute toward understanding the state of the diapause embryo and lay the basis and serve as a useful tool for further profound studies in the proteomics of Artemia at different developmental stages and physiological conditions.

Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation

To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis (F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection. (C) 2010 Elsevier Ltd. All rights reserved.

An etched porous interface for on-line capillary electrophoresis-based two-dimensional separation system

The construction and evaluation of an on-column etched fused-silica porous junction for on-line coupling of capillary isoelectric focusing (CIEF) with capillary zone electrophoresis (CZE) are described. Where two separation columns were integrated on a single piece of fused-silica capillary through the etched similar to4 to 5-mm length porous junction along the capillary. The junction is easily prepared by etching a short section of the capillary wall with HF after removing the polyimide coating. The etched section becomes a porous glass membrane that allows only small ions related to the background electrolyte to pass through when high voltage is applied across the separation capillary. The primary advantages of this novel porous junction interface over previous designs (in which the interface is usually formed by fracturing the capillary followed by connecting the two capillaries with a section of microdialysis hollow fiber membrane) are no dead volume, simplicity, and ruggedness, which is particularly well suited for an on-line coupling capillary electrophoresis-based multiple dimensional separation system. The performance of the 2D CIEF-CZE system constructed by such an etched porous junction was evaluated by the analyses of protein mixtures.

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregrarn is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.

Spatiotemporal heterogeneity of plankton communities in Lake Donghu, China, as revealed by PCR-denaturing gradient gel electrophoresis and its relation to biotic and abiotic factors

The 16S and 18S rRNA genes of planktonic organisms derived from five stations with nutrient gradients in Lake Donghu, China, were studied by PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, and the relationships between the genetic diversity of the plankton community and biotic/abiotic factors are discussed. The concentrations of total nitrogen (TN), total phosphorus (TP), NH4-N and As were found to be significantly related (P < 0.05) to morphological composition of the plankton community. Both chemical and morphological analyses suggested that temporal heterogeneity was comparatively higher than spatial heterogeneity in Lake Donghu. Although the morphological composition was not identical to the DGGE fingerprints in characterizing habitat similarity, the two strongest eutrophic stations (I and II) were always initially grouped into one cluster. Canonical correspondence analysis suggested that the factors strongly correlated with the first two ordination axes were seasonally different. The concentrations of TN and TP and the densities of rotifers and crustaceans were generally the main factors related to the DGGE patterns of the plankton communities. The study suggested that genetic diversity as depicted by metagenomic techniques (such as PCR-DGGE fingerprinting) is a promising tool for ecological study of plankton communities and that such techniques are likely to play an increasingly important role in assessing the environmental conditions of aquatic habitats.

Two-dimensional simulation of an electron cyclotron resonance plasma source with self-consistent power deposition

Using a refined two-dimensional hybrid-model with self-consistent microwave absorption, we have investigated the change of plasma parameters such as plasma density and ionization rate with the operating conditions. The dependence of the ion current density and ion energy and angle distribution function at the substrate surface vs. the radial position, pressure and microwave power were discussed. Results of our simulation can be compared qualitatively with many experimental measurements.

The cellular structure of a two-dimensional H2/O2/Ar detonation wave

In this paper, the cellular structure of a two-dimensional detonation wave in a low pressure H2/O2/Ar mixture calculated with a detailed chemical reaction model, high order scheme and high resolution grids is investigated. The regular cellular structure is produced about 1 ms after introducing perturbations in the reaction zone of a steady one-dimensional detonation wave. It is found from the present resolution study that the discrepancies concerning the structure type arising from the coarser grid employed can be resolved using a sufficiently fine grid size of 0.05 mm and below and shows a double-Mach-like strong-type configuration. During the structure evolution process, the structure configuration does not change much in the periods before and after the triple point collision. Through the triple point collision, three regular collision processes are observed and are followed by a quick change to the double-Mach-like configuration. The simulated structure tracks show that there are three different tracks associated with different triple points or the kink on the transverse wave. Comparisons with previous work and experiments indicate the presence of a strong structure for an ordinary detonation.

Characteristics and Sub-Characteristics of Two-Dimensional Parabolized Stability Equations

特征分析表明：对原始扰动量的抛物化稳定性方程组(PSE),它在亚超音速区分别具有椭圆和抛物特性,给出PSE特征对马赫数的依赖关系,阐明PSE仅把信息对流-扩散传播特性抛物化,而保留了信息对流-扰动传播特性,因此PSE应称为扩散抛物化稳定性方程(DPSE)。

The Structure and Evolution of a Two-Dimensional H2/O2/Ar Cellular Detonation

This paper reports on two-dimensional numerical simulation of cellular detonation wave in a / / mixture with low initial pressure using a detailed chemical reaction model and high order WENO scheme. Before the final equilibrium structure is produced, a fairly regular but still non-equilibrium mode is observed during the early stage of structure formation process. The numerically tracked detonation cells show that the cell size always adapts to the channel height such that the cell ratio is fairly independent of the grid sizes and initial and boundary conditions. During the structural evolution in a detonation cell, even as the simulated detonation wave characteristics suggest the presence of an ordinary detonation, the evolving instantaneous detonation state indicates a mainly underdriven state. As a considerable region of the gas mixture in a cell is observed to be ignited by the incident wave and transverse wave, it is further suggested that these two said waves play an essential role in the detonation propagation.

General Solution to Two-Dimensional Nonslipping JKR Model with a Pulling Force in an Arbitrary Direction

In this paper, a generalized JKR model is investigated, in which an elastic cylinder adhesively contacts with an elastic half space and the contact region is assumed to be perfect bonding. An external pulling force is acted on the cylinder in an arbitrary direction. The contact area changes during the pull-off process, which can be predicted using the dynamic Griffith energy balance criterion as the contact edge shifts. Full coupled solution with an oscillatory singularity is obtained and analyzed by numerical calculations. The effect of Dundurs' parameter on the pull-off process is analyzed, which shows that a nonoscillatory solution can approximate the general one under some conditions, i.e., larger pulling angle (pi/2 is the maximum value), smaller a/R or larger nondimensional parameter value of Delta gamma/E*R. Relations among the contact half width, the external pulling force and the pulling angle are used to determine the pull-off force and pull-off contact half width explicitly. All the results in the present paper as basic solutions are helpful and applicable for experimenters and engineers.