多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩后进入毛细管反相柱进行反相分离 ,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .     

大鼠海马的表达蛋白质组学实验研究

目的：用蛋白质组学方法初步分析大鼠海马蛋白质的表达。方法：提取大鼠海马蛋白质样品后,用双向凝胶电泳对其分离,经考马斯亮蓝染色后,产生大鼠海马蛋白质双向凝胶电泳图谱。从凝胶上切割分离的蛋白质,经胰蛋白酶胶内酶解,通过基质辅助激光解吸／电离飞行时间质谱（MALDI-TOF-MS）对酶解后的肽段进行分析。根据肽段质谱数据,经数据库（NCBI）检索,对蛋白质进行鉴定。结果：鉴定了37种具有明确功能的蛋白质,它们分别属于代谢酶、细胞骨架蛋白、热休克蛋白、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、神经元特异蛋白及神经胶质蛋白。另外,鉴定了3种未知功能蛋白。结论：为建立大鼠海马蛋白质组数据库提供必要的资料,为在大鼠模型上研究神经疾病发病机理奠定基础。     

蛋白质的肽质谱指纹图分析方法的优化

肽质谱指纹图分析是一种常用的蛋白质的鉴定方法.为了提高这种方法鉴定蛋白质时序列覆盖率和准确度,以6个标准蛋白质为分析样品,对几种不同的酶解肽段的浓缩、脱盐和点样方法进行了检验和优化.结果发现,将酶解肽段的浓缩体积控制在5μl以下和采用10mmolL柠檬酸铵缓冲液板上脱盐能提高蛋白质鉴定的准确度;在点样的时候,采用先点样品再点基质的方法能明显提高匹配肽段的个数和信噪比.这些优化的样品制备方法明显地提高了MALDITOF质谱肽质谱指纹图分析方法鉴定蛋白质的可靠性.     

鼠肝质膜蛋白质组研究的方法评估

细胞质膜是细胞中重要的细胞器, 在肝功能的发挥中具有非常重要的作用. 使用蔗糖密度梯度离心纯化细胞质膜, 通过电子显微镜观察和免疫印迹法检验膜的纯度. 结果显示, 与组织匀浆成分相比, 质膜富集了20倍, 线粒体的污染减少了约50%. 提取的蛋白质用二/一维凝胶电泳(2DE/1DE)分离、胰酶酶解、电喷雾四极杆飞行时间质谱(ESI-Q-TOF)和基质辅助激光解吸飞行时间串联质谱(MALDI-TOF-TOF)鉴定, 或者提取的蛋白质直接进行溶液内酶解、液相色谱串联电喷雾离子阱质谱(LC-LTQ)(鸟枪法)鉴定. 一共鉴定了547个非冗余蛋白质, 其中34%为质膜或质膜相关蛋白质. 优化和评估了质膜蛋白质组研究的方法, 且对鼠肝质膜蛋白质组进行了系统的分析.     

鼠肝质膜蛋白质组研究的方法评估

细胞质膜是细胞中重要的细胞器, 在肝功能的发挥中具有非常重要的作用. 使用蔗糖密度梯度离心纯化细胞质膜, 通过电子显微镜观察和免疫印迹法检验膜的纯度. 结果显示, 与组织匀浆成分相比, 质膜富集了20倍, 线粒体的污染减少了约50%. 提取的蛋白质用二/一维凝胶电泳(2DE/1DE)分离、胰酶酶解、电喷雾四极杆飞行时间质谱(ESI-Q-TOF)和基质辅助激光解吸飞行时间串联质谱(MALDI-TOF-TOF)鉴定, 或者提取的蛋白质直接进行溶液内酶解、液相色谱串联电喷雾离子阱质谱(LC-LTQ)(鸟枪法)鉴定. 一共鉴定了547个非冗余蛋白质, 其中34%为质膜或质膜相关蛋白质. 优化和评估了质膜蛋白质组研究的方法, 且对鼠肝质膜蛋白质组进行了系统的分析.     

Jurkat T细胞质膜蛋白组学研究初探

目的:分析Jurkat T细胞质膜蛋白质组成,并对这些质膜蛋白的生理学过程和功能进行初步分析,为进一步研究Jurkat T细胞膜蛋白功能奠定基础.方法:首先采用差异密度梯度离心法提取Jurkat T细胞膜蛋白,然后将提取出的膜蛋白根据分子量大小通过SDS-PAGE进行初步分离,再进一步将分离出的蛋白条带切下进行胶内酶解,酶解后的肤段通过液相-芯片-离子阱质谱技术进行鉴定和生物信息学分析,建立Jurkat T细胞质膜蛋白全谱图,并进一步通过GO(Gene Ontology)对这些质膜蛋白进行功能分析.结果:成功提取了Jurkat T细胞的膜总蛋白,并建立了Jurkat T细胞质膜蛋白全谱图,共鉴定出618个质膜蛋白,经GO注释分析,其中与结合功能相关的质膜蛋白有493个,与信号转导活性相关的有186个,具有酶催化活性的有166个,具有转运活性的有137个,有些还具有酶调节活性、结构分子活性或者运动活性等,功能尚不清楚的有49个.结论:通过差异密度梯度离心,结合一维SDS-PAGE和HPLC-CHIP-MS/MS,成功建立了Jurkat T细胞质膜蛋白全谱图,并通过GO注释,初步分析了这些蛋白的功能和生理学过程,为进一步研究Jurkat T细胞质膜蛋白的功能奠定了基础.     

蓝色温和凝胶双向电泳用于分析嗜盐菌质膜蛋白复合物

为了揭示细胞对盐胁迫渗透适应的分子机制,以新鉴定的中度嗜盐芽孢杆菌Bacillussp.I121为实验材料,分析了该嗜盐菌质膜上的盐胁迫响应蛋白.为此,通过蓝色温和凝胶双向电泳(BN/SDS-PAGE)对纯化的质膜组分进行了差异蛋白质组学研究.经MALDI-TOF/TOF质谱分析,鉴定了8个盐胁迫响应蛋白.盐胁迫诱导上调表达的蛋白质包括ABC型转运蛋白、3-磷酸甘油透性酶、嘧啶核苷转运蛋白和甲酸脱氢酶,下调表达的蛋白质包括琥珀酸脱氢酶(succinate dehydrogenase)铁硫亚基、黄素蛋白亚基、细胞色素b556亚基以及分子伴侣DnaJ的同源蛋白;酶活力测定结果表明胁迫条件下上述蛋白质的活性变化与表达量变化相一致.这些蛋白质中绝大多数属于高度疏水的跨膜蛋白,主要负责物质跨膜运输及能量代谢.上述结果表明,中度嗜盐菌Bacillus sp.I121可通过加快跨膜物质运输,同时抑制TCA循环完成盐胁迫条件下相容性溶质脯氨酸和四氢嘧啶的合成与积累.也进一步证明,蓝色温和凝胶双向电泳不仅可用于线粒体、叶绿体中蛋白质复合物的分析,也同样适用于细胞质膜上高度疏水蛋白复合物的比较研究.     

大鼠脑皮质表达蛋白质组学研究

文章用蛋白质组学方法初步分析大鼠脑皮质蛋白质的表达。提取大鼠脑皮质蛋白质,双向凝胶电泳分离,考马斯亮蓝染色,胰蛋白酶胶内酶解,用基质辅助激光解吸/电离飞行时间质谱对酶解后的肽段进行分析,根据肽质量指纹图谱,检索专业数据库(Swissprot),对蛋白质进行鉴定。鉴定出84个蛋白,分别属于代谢酶、细胞骨架蛋白、热休克蛋白、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、神经元特异蛋白及神经胶质蛋白等。文章结果丰富了大鼠脑皮质蛋白质组数据库,为在大鼠模型上研究神经疾病奠定了基础。     

糜蛋白酶提高膜蛋白质谱分析序列覆盖率的胶内消化方法

近年来,质谱技术在膜蛋白结构与功能研究中被广泛应用。由于膜蛋白的跨膜结构域含有大量疏水性氨基酸,常常导致液质串联质谱检测的序列覆盖率较低,从而限制了质谱技术在膜蛋白结构与功能研究中的应用。文中利用人的整合膜蛋白维生素K环氧化物还原酶为模型,优化胶内消化条件,建立了一种稳定提高膜蛋白质谱序列覆盖率的糜蛋白酶胶内消化方法。通过探索钙离子浓度、pH值和缓冲体系对序列覆盖率、检测特异肽段的总数和类型以及特异肽段大小的影响,发现在5–10 mmol/L钙离子浓度、pH 8.0–8.5的Tris-HCl缓冲液中,可以兼顾序列覆盖率和肽段的多样性。该方法可以使膜蛋白的质谱覆盖率达到80%以上,将在膜蛋白结构与功能、膜蛋白相互作用位点的鉴定以及膜蛋白与小分子药物结合位点的鉴定等研究中具有广泛的应用价值。     

一种适用于质谱分析的简化胶内酶解方法

在常规方法的基础上,设计了一种适于质谱分析的简化胶内酶解方法。改进的步骤包括:(1)通过加大洗涤所用超纯水量、延长涡混时间来强化凝胶洗涤的环节;(2)加入酸化处理来提高胰蛋白酶的活性;(3)在预酶解时不加CaCl_2,减少了酶的自切作用;(4)省略了蛋白质样品脱盐、脱SDS的步骤;(5)直接吸取酶解液进行质谱分析。系统比较该简化酶解法和最新报道的一种酶解方法的质谱鉴定效果,简化法能有效减少酶解后肽段的损失,增加质谱数据库搜索的信息量,得到更可靠的蛋白质鉴定结果。     

保护性氧化盐酸水解法分析蛋白质水解氨基酸

本文介绍了在保护剂存在下,通过过甲酸氧化,再以盐酸水解,在50分钟的分析循环时间内准确地分析17种氨基酸及三种含硫氨基酸衍生物的分析方法。     

Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion

Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.     

Rapid Identification of Viable Bacterial Spores Using a Fluorescence Method

A method has been devised to differentiate viable and nonviable bacterial spores. “Germination-like” changes are initiated in spores with performic acid and lysozyme. The germinated spores are stained with aqueous acridine orange, a fluorescent dye. Nonviable spores fluoresce lemon-green and viable spores orange-red. It is proposed that with the use of a membrane filter resistant to performic acid and lysozyme, the method may be used for spore enumeration in foods in about 4 hr compared to conventional plating methods, which usually require up to 72 hr.     

Determination of the Cysteine and Cystine Content of Proteins by Amino Acid Analysis: Application to the Characterization of Disulfide-Coupled Folding Intermediates

A method has been developed for the simultaneous detection of cysteine and cystine in proteins by amino acid analysis. In this method, the sulfhydryl groups of the cysteine residues are first blocked with 2-aminoethyl methanethiosulfonate (AEMTS). This reagent converts all free sulfhydryl groups to mixed disulfides with 2-aminoethanethiol (AET). The isolated blocked protein is subjected to oxidation with performic acid prior to hydrolysis and amino acid analysis. This procedure quantitatively converts the 2-aminoethanethiol blocking groups into taurine, and all cysteine residues (including those involved in disulfide bonds) into cysteic acid. Both of these derivatives are stable and can be recovered quantitatively by amino acid analysis. The speed and specificity with which AEMTS reacts with thiols make this method particularly effective for the characterization of disulfide-coupled folding intermediates.     

Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

Dependence of human somatotropin activity on interchain disulfide bridges

The importance of the disulfide bridges for human somatotropin activity is investigated. The activity of somatotropin is tested by the tibia, radioimmuno-, and radioligand assays. The cleavage of disulfide bridges by sulfitolysis, reduction with dithiothreitol, or oxidation with performic acid does not completely abolish hormone activity. There is only one exception: in the radioligand assay, oxidized somatotropin is not able to displace native somatotropin from rat liver membranes. The diminution of hormone activity is independent of the charges of the groups introduced to the cysteine residues. The radioimmuno- and radioligand assays are more sensitive to conformational alterations in the somatotropin molecule than the biological test system.     

用Beckman 121MB型氨基酸分析仪快速分析含硫氨基酸和色氨酸

蛋白质中胱氨酸、蛋氨酸和色氨酸在用盐酸水解时损失较大,常用过甲酸氧化法和碱水解法处理。采用文中方法分析时间短,分辨率高,半胱磺酸、甲硫氨酸砜和色氨酸的保留时间分别为５．３、１１．６和２０．４分钟。每分析一个样品需２６分钟,且基线平稳、重复性好、色谱图清晰。     

Versatile synthesis of α-substituted taurines from nitroolefins

A series of 1-substituted and 1,1-disubstituted taurines were synthesized from nitroolefins via the Michael addition with sodium ethylxanthate, oxidation with performic acid, and reduction with hydrogen in the presence of palladium on carbon powder. The current route is a versatile and salt-free method for synthesis of both aliphatic and aromatic 1-substituted and 1,1-disubstituted taurines.     

Deamination of protein lysyl ε-amino groups by peroxidase in vitro

Peroxidase, catechol, and hydrogen peroxide were shown to react with proteins, causing a decrease in lysine detectable after acid hydrolysis. The loss of lysine did not occur in the presence of benzenesulfinic acid which suggested that the quinones formed by peroxidase had oxidized some lysyl residues to lysyl aldehyde that formed a cyclic ninhydrin negative Shiff's base. When peroxidase treated protein was oxidized with performic acid prior to hydrolysis a new ninhydrin positive compound was found, which was shown by cochromatography and mass spectroscopy to be α-aminoadipic acid. The α-aminoadipic acid recovered accounted for (20–40)% of the lysine lost.     

An approach to the antigenic structure of arginine kinase and creatine kinase. Physical, chemical and immunological study of some modified derivatives.

The antigenic structure of arginine kinase and creatine kinase has been approached using chemical modifications and enzymatic cleavage. Mild performic oxidation that, with a restricted number of oxidized amino acid residues, results for both enzymes in a severe decrease of the helical structure and in a large increase of the protein-solvent interactions, affects differently their antigenic reactivity: when compared with antisera to the homologous native enzymes, arginine kinase and its oxidized derivative cross-react fully, while creatine kinase and its oxidized derivative cross-react only about 30%. The persistence of the antigenic reactivity of arginine kinase through drastic structural alterations is confirmed by the high inhibitory capacity (about 80%) of crude tryptic hydrolyzates towards the combination of argining kinase with its specific antibodies. Tryptic peptides of creatine kinase, obtained in the same conditions, inhibit weakly (about 12%) the homologous antigen-antibody interaction. The participation of the lysines in the antigenicity of arginine kinase and creatine kinase is suggested by the enhanced inhibitory capacity of the tryptic hydrolyzates when the cleavage is restricted to the arginyl peptide bounds, and was verified for arginine kinase through assays with lysine-modified derivatives.