基于OFFGEL预分离二维液质联用技术在大鼠海马膜蛋白中的应用

以大鼠海马膜组分为研究对象,以等电聚焦OFFGEL为研究手段,结合反相液相色谱质谱联用技术,考察对酶切肽段的分离富集性能,同时结合DAVID分析工具对分离富集得到的酸性蛋白质进行的GO分析.结果表明,海马膜组分的酶切肽段经过OFFGEL分离后,得到24个不同馏分,每个馏分经过纳流液相色谱-质谱检测,发现有77% 的肽段专属于一个馏分,在酸性范围内的肽段等电点偏差较小,可以达到良好的聚焦效果;酸性蛋白的GO分析结果显示,质膜和物质的跨膜运输所占比例最大.OFFGEL作为一种预分离手段,可以应用于生物样品的大规模蛋白鉴定,在进一步蛋白质层面的生物功能研究中表现出良好的应用前景.     

聚苯乙烯为致孔剂的分子印迹膜对溶菌酶吸附性能的研究

探索聚苯乙烯微球的添加对于分子印迹膜对溶菌酶(Lysozyme, Lyz)吸附性能的影响.以溶菌酶为模板分子,丙烯酰胺、N,N'-亚甲基双丙烯酰胺为功能单体、聚苯乙烯(Polystyrene,PS)微球为致孔剂,制备聚苯乙烯为致孔剂的分子印迹聚合物膜(PS-MIP).PS-MIP的吸附平衡时间为30 min,短于未添加聚苯乙烯微球的分子印迹聚合物膜(MIP);PS-MIP与MIP对于Lyz的吸附能力均明显好于聚苯乙烯为致孔剂的非分子印迹聚合物膜(PS-NIP)与未添加聚苯乙烯微球的非分子印迹聚合物膜(NIP);PS-MIP对Lyz的选择性要好于MIP,而卵清蛋白和牛血清白蛋白在PS-MIP和MIP上的荧光信号没有差别.实验结果表明,在分子印迹膜聚合过程中添加聚苯乙烯微球,有利于缩短吸附时间,增大溶菌酶的吸附量并提升选择性.     

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.     

Comparative proteomics analysis of <Emphasis Type="Italic">OsNAS1</Emphasis> transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> under salt stress

Salt stress is one of the major abiotic stresses in agricultural plants worldwide. We used proteomics to analyze the differential expression of proteins in transgenic OsNAS1 and non-transformant Brassica napus treated with 20 mmol/L Na₂CO₃. Total protein from the leaves was extracted and separated through a high-resolution and highly repetitive two-dimensional electrophoresis (2-DE) technology system. Twelve protein spots were reproducibly observed to be upregulated by more than 2-fold between transgenic and non-transformant B. napus. These 12 spots were digested in-gel with trypsin and characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain the peptide mass fingerprints. Protein database searching revealed that 5 of these proteins are involved in salt tolerance: dehydrogenase, glutathione S-transferase, peroxidase, 20S proteasome beta subunit, and ribulose-1,5-bisphosphate carboxylase/oxygenase. The potential functions of these identified proteins in substance and energy metabolism, stress tolerance, protein degradation, and cell defense are discussed. The salt tolerance of the transgenic rapeseed was significantly improved by the introduction of the OsNAS1 gene from Brazilian upland rice of Oryza sativa (cv. IAPAR 9).     

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.     

DMSO处理后中国仓鼠卵巢细胞的蛋白质组变化分析

在培养基中添加二甲基亚砜(DMSO)可使基因工程中国仓鼠卵巢细胞(CHO)外源蛋白乙肝表面抗原(HBsAg)的比生产速率提高4倍以上.为了进一步研究DMSO的作用机理,采用双相凝胶电泳技术分离在培养基中添加1.5%的DMSO后CHO细胞的总蛋白,胶态考马斯亮蓝G-250染色,GS-800凝胶扫描仪获取凝胶图像,并用PDQUEST软件分析,测得每张图谱平均斑点数量为1852±123,以其中一块胶为参考胶进行4块胶间的斑点匹配,其平均匹配的点数达1326±100,平均匹配率为71.6%以上.通过分析得到18个蛋白质点在添加了DMSO后表达量有明显变化(其中12个蛋白表达量明显降低,6个蛋白表达量明显增加.用基质辅助激光解析电离飞行时间质谱测定其胶内酶解后的肽质指纹谱图和串级质谱图,用GPS软件查询NCBI数据库进行了鉴定.鉴定结果表明,这些差异蛋白中一些是与细胞周期有关的蛋白,一些是与细胞能量代谢相关的蛋白,还有一些是与蛋白翻译后修饰有关的酶,这些结果进一步深化了对DMSO提高CHO细胞表达HBsAg的机理的理解.     

东方蜜蜂微孢子虫30kD蛋白的LC-MS／MS质谱鉴定及分析

在微孢子虫（Microsporidia）侵染宿主的过程中,与侵染相关的蛋白主要分布在30kD左右。本研究分别采用煮沸法、不同浓度碱处理发芽法提取东方蜜蜂微孢子虫（Nosema ceranae）蛋白并通过SDS-PAGE电泳进行比较,发现0．1mol／LKHCO3、K2CO3混合液（pH值为10．7）发芽法提取出的蛋白条带更丰富。回收30kD左右蛋白进行LC-Ms／Ms质谱测定并检索蜜蜂微孢子虫全基因组预测蛋白数据库,共鉴定获得的269个预测蛋白,匹配COG数据库后进行了蛋白功能分类。结果表明30kD蛋白主要包括翻译转录蛋白、核糖体结构蛋白、修饰蛋白、转化蛋白及分子伴侣等。从中还鉴定到与侵染相关的7种潜在孢壁蛋白（Spore wall protein,SWP）和3种极管蛋白（Polar tube protein,PTP）,并对之进行蛋白基因的序列分析,进而发现注释到的PTP1、PTP2和家蚕微孢子虫的PTP1、PTP2具有明显的共线性,其中SWP12的变异度相对较小;结合多重序列比对结果,表明SWP12在东方蜜蜂微孢子虫中是一类较保守的孢壁蛋白。本研究对于蜜蜂微孢子虫蛋白的提取方法比较和侵染相关的后选靶标蛋白的确定具有重要的参考意义。     

适合于MALDI-TOF质谱分析蛋白质及亚基的SDS-PAGE技术

介绍适合于MALDI TOF质谱分析蛋白质及亚基的SDS PAGE技术,其主要特点是采用铜染色法在凝胶板上直接显色且采用有机溶剂萃取电泳纯的蛋白质和亚基,并用MALDI TOF质谱技术直接分析它们的分子量.本技术具有简便、快速和损失率小等优点,尤其适合于蛋白质组学、利用数据库分析蛋白质同源性和确定蛋白质种类等结构信息的研究,是一种较为理想的SDS PAGE和MALDI TOF质谱非在线联用分析技术.     

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.     

A proteomic analysis of bacterial strain<Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> RT19 subjected to salt shock

Sinorhizobium fredii RT19, a strain of freeliving bacteria, was subjected to salt shock and its protein expression profiles were analyzed by differential display proteome approaches. The results of separation by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) showed that the number of resolved proteins was 481, 465 and 424, corresponding to salt-free control, 5 and 50 min 1 mol/L salt treatment, respectively. Among the resolved proteins, 82 in total had altered expression in response to salt-shock stress. 26 out of the 82 proteins were induced and 23 were completely inhibited, while 12 were up-regulated and 21 down-regulated in response to salt shock. In addition, the appearance of differentially displayed proteins responding to different salt shock periods is also reported. The identity of the 26 induced proteins was revealed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) followed by database searching. Among them, 20 were assigned to proteins with known functions. Their roles in response to salt shock stress are discussed.     

利用双向电泳-质谱技术鉴定牦牛和犏牛睾丸中 的差异表达蛋白质

比较了牦牛和犏牛睾丸组织中蛋白质的表达差异; 应用双向电泳-质谱技术, 对牦牛和犏牛的睾丸组织中差异蛋白质进行分离和鉴定; 对获得的22个差异表达点进行质谱鉴定和数据库搜索, 获得了19个蛋白质, 其中有3种分子伴侣可能与犏牛雄性不育有关; 研究结果表明, 牦牛和犏牛睾丸组织中存在差异表达蛋白, 并可利用双向凝胶电泳进行分离, 为进一步分析犏牛雄性不育的原因提供了线索.     

利用双向电泳-质谱技术鉴定牦牛和犏牛睾丸中的差异表达蛋白质

比较了牦牛和犏牛睾丸组织中蛋白质的表达差异;应用双向电泳-质谱技术,对牦牛和犏牛的睾丸组织中差异蛋白质进行分离和鉴定;对获得的22个差异表达点进行质谱鉴定和数据库搜索,获得了19个蛋白质,其中有3种分子伴侣可能与犏牛雄性不育有关;研究结果表明,牦牛和犏牛睾丸组织中存在差异表达蛋白,并可利用双向凝胶电泳进行分离,为进一步分析犏牛雄性不育的原因提供了线索.     

白斑综合征中国对虾肝胰腺蛋白质组学研究的技术探索

建立白斑综合征中国对虾肝胰腺蛋白质组学技术体系,以患白斑综合征中国对虾的肝胰腺为研究对象,健康中国对虾肝胰腺为对照组,探索2种蛋白质组学差异蛋白的分离和鉴定的方法. 采用双向凝胶电泳(4～7固相pH梯度干胶条,10%SDS-PAGE)分离蛋白质组,银染显色,图像分析软件比较分析,识别差异蛋白,胶上扣点,处理后通过基质辅助激光解析电离飞行时间质谱（MOLDI-TOF）得到肽质量指纹图谱,软件分析后应用Mascot数据库搜索软件鉴定蛋白. 分别获得中国对虾患白斑病组及对照组正常肝胰腺蛋白质组表达图谱. 分别识别出203和107个蛋白质点. 筛选并尝试鉴定数个差异蛋白. 对各方法步骤进行了技术探讨,对实验过程提出了一系列建议. 建立了双向电泳及生物质谱方法研究中国对虾白斑病的蛋白质组学技术体系. 该体系拓宽了对虾免疫生理研究的途径,以从蛋白质水平探讨对虾发病机制,寻找有效药物靶点.     

白酒中微量芳香成分的气相色谱-质谱检测

分别采用DB-WAX、HP-5MS石英毛细管柱,选用直接进样方式,利用气相色谱-质谱联用仪对重庆某白酒厂4种白酒中的微量成分进行定性分析,通过图谱库检索,鉴定出4种白酒中微量芳香物质的成分,并研究了其在不同白酒中的差异.本方法为白酒的勾兑和质量控制提供了一种高效、准确的分析方法.     

巴西橡胶树树皮蛋白质组学分析体系的构建

巴西橡胶树(Hevea brasiliensis)是一种重要的产胶植物,其体内合成和贮存胶乳的地方是乳管系统,而乳管主要分布于树皮中。以10年生巴西橡胶树无性系“热研88-13”为研究对象,采用改进的三氯乙酸(TCA) 丙酮沉淀法提取树皮蛋白,使用17 cm,pH 4~7的IPG胶条进行双向电泳,得到良好的蛋白图谱。PDQuest软件(Bio rad)分析银染后的凝胶,共得到约350个蛋白点。从这些蛋白点中随机选取10个蛋白点经基质辅助激光解析电离飞行时间质谱(MALDI TOF MS)分析后,搜索NCBInr数据库对蛋白进行鉴定。结果显示,这些蛋白主要包括两种来源于巴西橡胶树中的重要蛋白,即橡胶小粒子蛋白(Small rubber particle protein)和橡胶树凝集因子(hevein),此外还有一些其他功能和未知功能的蛋白。     

对B.t毒素敏感与抗性的昆虫细胞的差异蛋白质肽质指纹分析

采用2-DE技术分析了粉纹夜蛾TnH5对B.t CrglAC毒素敏感和抗性2种细胞总蛋白质,建立了TnH5细胞双向电泳图谱,获得了一些差异蛋白质.对部分差异蛋白质点采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/MS)测定了其胶内酶解后的肽质指纹图谱(PMF),查询数据库,结果表明,部分差异斑点分别与胞质外周蛋白、脂多糖生物合成蛋白、一种脱氢酶和类免疫球蛋白等类似.     

红莲型水稻二核期花粉蛋白质组学分析

对红莲型细胞质雄性不育水稻的不育系、保持系和杂种F1二核期花粉总蛋白质采用固相pH梯度等电聚焦/SDS-PAGE进行了双向电泳分离,获得了分辨率和重复性较好的双向电泳图谱．对其中15差异点进行了肽质指纹图分析或者LC-MS/MS分析．对已鉴定的蛋白质点进行亚细胞定位和功能分析,发现不育系相对于可育系有部分参与蛋白质缺失或表达量降低,可能与线粒体提供能量不足而导致的花粉不能正常发育有关．     

用双向电泳-质谱初步分析大熊猫初乳和常乳中差异表达的乳清蛋白

目的 是分析大熊猫初乳和常乳中差异表达的乳清蛋白.利用双向电泳-质谱技术首次对一只大熊猫产后第1d和第22 d的乳清蛋白进行初步的分离与鉴定,结果成功得到11种差异表达的蛋白质,值得注意的是,其中有3种蛋白质与免疫、抗菌有关,包括抗白细胞蛋白酶、溶菌酶C,C反应蛋白.推测这3种蛋白质可能参与大熊猫乳腺或幼仔的防御功能.研究结果表明,用双向电泳-质谱技术可以分析大熊猫初乳和常乳中的差异表达蛋白质,有助于认识大熊猫乳的组成和功能特性.     

Proteomic analysis of a toxic dinoflagellate Alexandrium catenella under different growth phases and conditions

Alexandrium is a widely spread dinoflagellate genus throughout many regions of the world,which not only causes the harmful algal blooms(HABs) but also results in the paralytic shellfish poisoning(PSP) throughout the world.This study compared protein profiles of A.catenella grown under different growth phases and conditions using a proteomic approach,and identified the differentially expressed proteins.The results showed that the expressions of proteins identified in three different regions of the gels,the groups 1,2 and 3 proteins,varied significantly with the growth phases and conditions.Group 1 proteins and six Group 2 proteins were highly expressed at the initial,exponential and stationary growth phases,eight Group 2 proteins were highly expressed only at the initial phase,and Group 3 proteins were highly expressed at the exponential and/or stationary phases.However,all these proteins were expressed at low levels or were barely visible at the dissipation phase.The expressions of groups 1 and 2 proteins were low or barely visible in various growth conditions except in continuous darkness they were highly expressed.Group 3 proteins,on the other hand,were overexpressed in continuous illumination and expressed at low levels or barely visible in continuous darkness or under nitrate-starvation.The data from MALDI-TOF-TOF mass spectrometry demonstrated that these differentially expressed proteins were associated with macromolecular biosynthesis,photosynthesis,tRNA synthesis and DNA stability,stress response and cell division regulation.Synthetase was the major component of the altered proteins.This is one of the first comprehensive proteomic study of a dinoflagellate,A.catenella,that provides a fundamental understanding of the proteins involved in A.catenella growth and response to environmental stresses,and potential physiological indicator proteins related to growth and environmental stress have been identified.     

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。