Proteomics of a toxic dinoflagellate Alexandrium catenella DH01: Detection and identification of cell surface proteins using fluorescent labeling

Alexandrium catenella DH01 is a toxic dinoflagellate species that is able to not only produce paralytic shellfish toxins,but also cause harmful algal blooms along the coast of China.In this study,we presented a new protocol for specific labeling and detection of the cell surface proteins(CSPs) of A.catenella DH01 cells using CyDye difference gel electrophoresis(DIGE) fluor minimal dyes.CSPs were identified using two-dimensional gel electrophoresis(2-DE) and MALDI TOF-TOF mass spectrometry(MS).The results showed that the fluorescent cyanine dye Cy3 could specifically label the CSPs of A.catenella DH01,with minimal labeling of intracellular proteins.Among three protein extraction methods evaluated,the Trizol method was the most efficient to extract CSPs with respect to protein spot number and resolution.Forty-one CSPs were separated and identified from A.catenella DH01 by 2-DE,in which 14 were identified in the protein database using MALDI TOF-TOF MS analysis.This work represents the first attempt to investigate the CSPs of A.catenella using the CyDye DIGE fluor dyeing method that provides a potentially important tool for future comprehensive characterization of CSPs and elucidation of the physiological functions of CSPs in dinoflagellates.     

Proteomics study of the effects of high pigment-1 on plastid differentiation during the ripening of tomato fruits

The chloroplast-to-chromoplast difference is a highly regulated process. Tomato （Solanum lycopersicum） high-pigment mutant hp-1 shows an enhanced accumula- tion of carotenoids coupled with an increase in the volume and number of plastids. However, how chromoplasts of hp-] acquire their specific metabolic characteristics is still unclear. A comparison of proteome profiles from plastids at the mature green, breaker, and red stages of tomato fruits showed 45 differentially expressed proteins. These identi- fied proteins fell into six different functional categories. Our results showed that most of the proteins related to the Calvin cycle increased transitorily only at the early breaker stage, and remained unchanged at the early red stage. We found 18 proteins that were differentially expressed between the wild type and hp-1. The abundance of GCPE in chromoplasts of hp-1 was higher than that in the wild type. Meanwhile, we found that heat shock proteins were only present specifically in the mature green stage chromoplasts of hp-1 but not in the wild type. This suggested that GCPE and heat shock proteins might play important roles in the accumulation of high carotenoids in hp-1. Overall, our results could be helpful for understanding chloroplast-to-chromoplast differentiation and the function of hp-1.     

应用cDNA微阵列芯片筛选胃癌转移相关基因的初步研究

采用cDNA微阵列技术建立胃癌原发灶和淋巴结转移灶基因表达谱,识别和克隆胃癌转移相关基因。用含10000个已知基因和7000个EST的cDNA微阵列分析胃癌原发灶和淋巴结转移灶表达谱的变化,利用生物信息学分析差异表达基因, RT-PCR和反向Northern点杂交验证cDNA微阵列结果。发现2倍以上的差异表达基因601个,其中淋巴结转移灶中表达上调527个,表达下调74个;2倍以上的差异EST 71个,其中淋巴转移灶中表达上调62个,表达下调9个。在胃癌原发灶中,与细胞免疫、发育、信号转导功能相关基因存在高表达;而在淋巴结转移灶中,与细胞生长、细胞周期、细胞运动和粘附功能相关基因存在高表达。RT-PCR和反向Northern点杂交结果进一步证实carbonic anhydraseⅡ、IGFBP-4基因高表达与胃癌转移相关。通过分析胃癌原发灶和淋巴结转移灶表达谱的变化,发现一些与胃癌转移相关的基因和EST,为进一步寻找和克隆胃癌转移相关基因提供研究线索。     

基于双向电泳技术的丙酮丁醇梭菌ATCC824不同产物阶段膜蛋白质组比较

建立丙酮丁醇梭菌在产酸阶段和产有机溶剂阶段的膜蛋白质组;并进行比较分析。研究采用差速离心法提取了该菌在产酸阶段和产有机溶剂阶段的膜蛋白,进行双向电泳分离后,对差异蛋白进行质谱分析。不同生长阶段膜蛋白质组存在明显差异,经质谱鉴定发现9个差异蛋白质,其中3个在产酸阶段高表达,6个在产有机溶剂阶段高表达。生物信息学分析表明,大部分蛋白含有明确的膜定位信息。产酸阶段高表达蛋白多与细胞信号转导有关,而产有机溶剂阶段高表达蛋白功能分布相对复杂。丙酮丁醇梭菌在不同产物阶段的差异膜蛋白被成功鉴定,它们可能在不同产物阶段的转换和细胞信号转导等过程中起到重要调控作用。     

SELDI-TOF-MS in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease(COPD) has a profound impact on daily life,yet remains underdiagnosed and undertreated.This study aims to discover potential protein biomarkers for diagnosis and classification of COPD.Fifty-seven COPD patients and 40 controls were divided into a training set(30 COPD patients,20 healthy controls) and a test set(27 COPD patients,20 healthy controls).Serum proteomic profiles were measured using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS).A classification tree was established using Biomarker Pattern Software(BPS).Next we screened distinct proteins present in patients with acute exacerbations of COPD(AECOPD),stable COPD and healthy controls,in order to establish diagnostic models for classification of COPD.Twenty peaks showed statistically significant differences between COPD patients and healthy controls(P < 0.05).Two proteomic peaks(3167 and 5477 m/z) were chosen by BPS to establish a classification tree in the training set.The sensitivity and specificity of this classification tree were 92.59% and 90.00% respectively in the testing set.Furthermore,differently expressed proteins were detected among the patients with AECOPD,stable COPD,and healthy controls.Two protein profiles(3167 and 4645 m/z) could distinguish between stable COPD patients and healthy controls.Three protein profiles(3167,2963 and 2973 m/z) could distinguish between AECOPD patients and healthy controls.Three protein profiles(5476,14039 and 2831 m/z) could distinguish between stable COPD patients and AECOPD patients.SELDI-TOFMS Proteinchip technology is a quick,easy and practical,high throughput analytic method.It shows the diagnostic models established by distinguished proteomic peaks can discriminate COPD patients from healthy control and can identify different stages of COPD.It will provide a highly accurate approach for diagnosis and clinical staging of COPD.     

高效液相色谱中的蛋白质手性固定相

对近年来报道的各种高效液相色谱蛋白质手性固定相的研究进行了综述.主要介绍了蛋白质手性固定相的制备、分类、组成、性质、拆分机理及其应用,讨论了各类蛋白质手性固定相的优缺点,指出了目前存在的问题和今后的研究方向.     

A data-mining approach to biomarker identification from protein profiles using discrete stationary wavelet transform

Objective： To develop a new bioinformatic tool based on a data-mining approach for extraction of the most informative proteins that could be used to find the potential biomarkers for the detection of cancer. Methods： Two independent datasets from serum samples of 253 ovarian cancer and 167 breast cancer patients were used. The samples were examined by surfaceenhanced laser desorption/ionization time-of-flight mass spectrometry （SELDI-TOF MS）. The datasets were used to extract the informative proteins using a data-mining method in the discrete stationary wavelet transform domain. As a dimensionality reduction procedure, the hard thresholding method was applied to reduce the number of wavelet coefficients. Also, a distance measure was used to select the most discriminative coefficients. To find the potential biomarkers using the selected wavelet coefficients, we applied the inverse discrete stationary wavelet transform combined with a two-sided t-test. Results： From the ovarian cancer dataset, a set of five proteins were detected as potential biomarkers that could be used to identify the cancer patients from the healthy cases with accuracy, sensitivity, and specificity of 100%. Also, from the breast cancer dataset, a set of eight proteins were found as the potential biomarkers that could separate the healthy cases from the cancer patients with accuracy of 98.26%, sensitivity of 100%, and specificity of 95.6%. Conclusion： The results have shown that the new bioinformatic tool can be used in combination with the high-throughput proteomic data such as SELDI-TOF MS to find the potential biomarkers with high discriminative power.     

Des(rhamnosyl) verbascoside抗HBV作用的定量蛋白质组学研究

为探究des(rhamnosyl) verbascoside体外抗乙型肝炎病毒(HBV)的活性作用和机制,本研究以des(rhamnosyl) verbascoside为实验药物对HepG2.2.15细胞进行干预,实验分为药物干预组和对照组,采用串联质谱标签(Tandem Mass Tag, TMT)蛋白质组学方法对提取的总蛋白进行分析。结果表明,共筛选得到300个差异表达蛋白,其中有109个上调蛋白,191个下调蛋白。基因本体论(Gene Ontology, GO)分析结果显示,差异蛋白主要参与DNA复制(DNA replication)、鞘糖脂代谢(Glycosphingolipid metabolic process)、细胞增殖(Cell proliferation)、寡糖分解代谢(Oligosaccharide catabolic process)等生物学过程,以及DNA聚合酶活性(DNA polymerase activity)、丝氨酸型羧肽酶活性(Serine-type carboxypeptidase activity)、DNA引物酶活性(DNA primase acti...     

金黄色葡萄球菌细胞内生物大分子含量变化的光镊拉曼光谱研究

【目的】利用光镊拉曼光谱技术(Laser Tweezers Raman Spectroscopy,LTRS)研究金黄色葡萄球菌(Staphylococcus aureus)细胞内主要生物大分子含量的动态变化,探寻监测微生物代谢状态变化的有效工具。【方法】收集分批培养过程不同时间点金黄色葡萄球菌细胞的拉曼光谱,观察其特征峰依赖时间的变化特点。【结果】归属于核酸的特征峰,如667cm~(-1)峰,724cm~(-1)峰,781cm~(-1)峰,809cm~(-1)峰和1 574cm~(-1)峰在整个分批生长阶段持续降低,在对数生长期下降幅度尤其剧烈,反映了对数生长期细胞分裂快速,DNA复制活跃;与蛋白质相关的特征峰,如850cm~(-1)峰,1 001cm~(-1)峰和1 662cm~(-1)峰,在对数生长期显著增强,而到稳定期峰强度基本维持恒定。【结论】光镊拉曼光谱技术能实时监测生物样品内生物大分子的含量,是单细胞水平上研究微生物代谢状态变化的有效工具。     

Proteome changes in the plasma of Papilio xuthus (Lepidoptera: Papilionidae): effect of parasitization by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

Although the biochemical dissection of parasitoid-host interactions is becoming well characterized, the molecular knowledge concerning them is minimal. In order to understand the molecular bases of the host immune response to parasitoid attack, we explored the response of Papilio xuthus parasitized by the endoparasitic wasp Pteromalus puparum using proteomic approach. By examining the differential expression of plasma proteins in the parasitized and unparasitized host pupae by two-dimensional (2D) electrophoresis, 16 proteins were found to vary in relation to parasitization compared with unparasitized control samples. All of them were submitted to identification by mass spectrometry coupled with a database search. The modulated proteins were found to fall into the following functional groups: humoral or cellular immunity, detoxification, energy metabolism, and others. This study contributes insights into the molecular mechanism of the relationships between parasitoids and their host insects.     

短日照处理诱导油松容器苗针叶差异表达蛋白质的功能分析

【目的】基于蛋白质水平探讨短日照处理调控油松(Pinus tabulaeformis)容器苗基础代谢和抗逆性机理的研究,完善油松容器苗的育苗和造林技术,为短日照处理育苗技术在我国北方地区困难立地造林中推广应用提供理论依据。【方法】以经日照长度为10 h、持续3 周短日照处理的油松容器苗针叶为研究对象,采用改良的酚法提取针叶蛋白。应用双向电泳结合二级质谱分析的蛋白质组学技术,研究短日照处理诱导油松容器苗针叶蛋白质表达的变化,分析差异表达蛋白质的鉴定和功能。【结果】成功获取5个短日照处理诱导表达差异显著的蛋白质,上调表达的是叶绿体放氧增强蛋白1(oxygen-evolving enhancer protein 1, OEE1)(蛋白点404)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)(蛋白点539)、延伸因子-Tu(elongation factor-Tu, EF-Tu)(蛋白点654)、核酮糖-1,5-二磷酸羧化酶/加氧酶活化酶(ribulose bisphosphate carboxylase/oxygenase activase, RCA)(蛋白点681); 下调表达的是磷酸甘油酸激酶1(phosphoglycerate kinase 1, PGK1)(蛋白点641)。这些蛋白质的功能主要涉及光合代谢、糖代谢和胁迫防御。【结论】短日照处理诱导油松容器苗蛋白质差异表达,对油松容器苗的基础代谢和抗逆性产生重要影响。     

Interaction of cellular-localized signature modules in response to prostate cancer

Rapid progress in high-throughput biotechnologies (e.g. microarrays) and exponential accumulation of gene functional knowledge makes it promising for systematic understanding of complex human diseases at the functional modules level. Current modular categorizations can be defined and selected more specifically and precisely in terms of both biological processes and cellular locations, aiming at uncovering the modular molecular networks highly relevant to cancers. Based on Gene Ontology, we identifed the functional modules enriched with differentially expressed genes and characterized by biological processes and specific cellular locations. Then, according to the ranking of the disease discriminating abilities of the pre-selected functional modules, we further defined and filtered signature modules which have higher relevance to the cancer under study. Applications of the proposed method to the analysis of a prostate cancer dataset revealed insightful biological modules.     

Interaction of cellular-localized signature modules in response to prostate cancer

Rapid progress in high-throughput biotechnologies (e.g. microarrays) and exponential accumulation of gene functional knowledge makes it promising for systematic understanding of complex human diseases at the functional modules level. Current modular categorizations can be defined and selected more specifically and precisely in terms of both biological processes and cellular locations, aiming at uncovering the modular molecular networks highly relevant to cancers. Based on Gene Ontology, we identifed the functional modules enriched with differentially expressed genes and characterized by biological processes and specific cellular locations. Then, according to the ranking of the disease discriminating abilities of the pre-selected functional modules, we further defined and filtered signature modules which have higher relevance to the cancer under study. Applications of the proposed method to the analysis of a prostate cancer dataset revealed insightful biological modules.     

Comparative proteomic analysis of the response in resistant and susceptible maize inbred lines to infection by Curvularia lunata

Proteins differentially expressed from maize leaves in response to the infection by Curvularia lunata strain CX-3 were identified through a high-resolution two-dimensional gel electrophoresis (2-DE) method. Two inbred lines, 78599-1 and E28, were used, respectively, as resistant and susceptible lines to CX-3 infection. Proteins were extracted from the fourth leaves of six- or seven-leaf stage plants sampled at 24, 36, 48, 60, and 72 h after inoculation with CX-3. Twenty-seven differentially expressed protein spots resolved on the 2-DE gels were identified by MALDI-TOF MS/MS. The results showed that these proteins are associated with photosynthesis, respiration,oxidative and drought stress tolerance as well as signal transduction in maize. Among stress-related proteins, the 22 kDa drought-inducible protein, putative glutathione peroxidase (GPX), and translation initiation factor (eIF-5A) were up-regulated in the resistant inbred line and were implicated in host defense response to C. lunata infection. It suggests that drought-inducible and oxidation stress-related proteins might directly contribute to maize resistance to C. lunata.     

亮氨酸类离子液体的合成及其气相色谱分离性能研究

研究亮氨酸类离子液体作为气相色谱固定相的分离性能及其应用.合成了L-亮氨酸乙酯四氟硼酸盐和L-亮氨酸乙酯双（三氟甲基磺酰）亚胺盐离子液体,将其作为固定相制备毛细管气相色谱柱,建立了50,80和110 ℃时的Abraham溶剂化参数模型,选用Grob试剂和正构烷烃混合物对固定相的气相色谱分离能力和选择性进行了考察.结果表明,亮氨酸类离子液体固定相具有良好的涂渍性能,对化合物进行保留所产生的多种相互作用力中,偶极相互作用力和氢键相互作用力占主导地位,具备较好的色谱分离能力和应用价值.      

美国山核桃不定根形成过程中相关蛋白质 的鉴定及功能分析

美国山核桃是扦插后极难生根的树种,为研究其不定根生根机制,利用MALDI-TOF-MS技术对美国山核桃不定根发育关键时期的差异蛋白进行鉴定分析,发现了48个表达相对差异的蛋白点,选择其中18个差异蛋白进行肽质谱指纹分析并进行同源性比较。结果表明,美国山核桃不定根形成的生理过程中产生了差异蛋白质,其中包括了能量代谢相关蛋白、逆境胁迫相关蛋白和信号传递相关蛋白等差异蛋白。分析可知:能量代谢相关蛋白有ATP合成相关酶类、光合作用相关蛋白及烯醇化酶等; 逆境胁迫相关蛋白与热激蛋白同源; 信号传递相关蛋白主要为细胞色素酶类和血红结合蛋白。     

水稻幼苗叶片应答稻瘟病侵染的差异蛋白谱分析

为了探讨水稻感病基因型种质响应稻瘟病侵染的蛋白质表达谱的变化规律和作用途径,以水稻感稻瘟病种质日本晴为材料,采用接种稻瘟病菌分生抱子悬浮液,24,48和72 h后提取叶片蛋白质,采用i TRAQ蛋白质组学技术研究稻瘟病胁迫下水稻叶片蛋白质组的变化.结果表明,稻瘟病侵染诱导了水稻幼苗叶片内涉及氧化还原平衡、防御、信号传导、糖和能量代谢、氨基酸代谢、光合作用,以及蛋白质代谢等代谢途径相关的53个蛋白质的表达量发生了改变.GO分析表明稻瘟病主要调控了植株体内细胞内平衡、代谢过程和蛋白质代谢等生物学过程.稻瘟病侵染激活了活性氧代谢、防御,以及热休克蛋白等相关的途径,而抑制了蛋白质生物合成过程.结合这些差异表达蛋白的丰度变化结合它们可能的功能,描绘了水稻应答稻瘟病侵染的蛋白质代谢网络,有助于在蛋白质水平上了解其应答过程.