大鳞大马哈鱼垂体促甲状腺素和促性腺激素分离纯化的研究

本文报道应用ConA-Sepharose 4B.亲和层析、凝胶过滤层析、离子交换层析及免疫亲和层析等技术,首次从大鳞大马哈鱼垂体中分离纯化了促甲状腺素(sTSH)和促性腺激素(sGTH)。在TSH生物测定中,纯化的。sTSH制品具有明显促进虹鳟幼鱼甲状腺体内分泌甲状腺素(T4)的生物活性,而sGTH无此活性。在GTH生物测定中,制备的sGTH具有显著诱导虹鳟卵母细胞体外培养成熟(GVBD)及分泌17α、20β-二氢孕酮的能力,而sTSH无此活性。HPLC表明sTSH和sGTH的分子量分别为27500和38000道尔顿。作者用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦检测了激素的纯度及等电点,用RIA或ELISA方法测定了几种垂体激素在sTSH终产物中的污染程度。     

基于电子捕获裂解/电子转运裂解串联质谱技术的蛋白质组学研究

蛋白质组学的兴起带动了质谱技术的快速发展,而质谱技术的进步则拓宽了蛋白质组学研究问题的广度.最近10年内,肽段或完整蛋白质在质谱仪中的裂解技术——电子捕获裂解(electron capture dissociation,ECD)与电子转运裂解(electron transfer dissociation,ETD)逐渐发展起来.ECD和ETD在蛋白质组学中的应用,特别是在蛋白质的翻译后修饰鉴定和自顶而下(Top-down)的完整蛋白质裂解研究中已经展示出了诱人的前景.对ECD和ETD的基本原理、质谱特点、仪器实现、数据解析算法与软件开发,以及在蛋白质组学中的应用进展等方面进行了比较系统全面的阐述,并对当前的研究问题、面临的技术挑战与未来的发展趋势等方面作了深入剖析.     

蛋白质组学及其在农业上的应用

论述了蛋白质组学与基因组学的关系,蛋白质组学的定义、功能、分类及其三大主要技术.详细评述了蛋白质组学技术在农业科学研究中的应用,如叶绿体蛋白质组,农作物与细菌的共生现象、植物叶绿体蛋白质组,作物抗旱性和雄性不育性等.最后展望了蛋白质组学这一生命科学中最新方法在农业科学中的应用前景.     

脂蛋白质组学研究进展

脂蛋白质组学(lipoproteomics)是一门应用蛋白质组学技术对脂蛋白(lipoprotein)进行全面、系统地分析和鉴定,进而了解脂蛋白的组成和功能以及与相关疾病发生、发展之间关系的新兴学科.近些年来,脂蛋白质组学研究促进了脂蛋白中蛋白质组分的急性期响应、补体激活、免疫响应、炎症响应、蛋白酶抑制等新功能的发现,显示了广阔的应用前景.本文对脂蛋白的功能和分类,以及目前应用于脂蛋白质组学研究的脂蛋白分离方法和蛋白质鉴定方法进行了简述,并综述了高密度脂蛋白、低密度脂蛋白和极低密度脂蛋白的脂蛋白质组学研究,及其在冠状动脉疾病、糖尿病、类风湿性关节炎等疾病研究的最新进展.     

蛋白质组学技术及其在药物成瘾研究中的应用

概述了蛋白质组学技术中分离、鉴定和定量方法及其在致瘾性药物(如酒精、烟碱、吗啡、可卡因和安非他命)研究中的应用。蛋白质组学技术是继后基因组学技术的一大重要突破。随着蛋白质分离、鉴定及定量技术的进步,尤其是覆盖范围广、分离效率高的液相色谱技术和高灵敏度质谱技术的发展,使得蛋白质组学越来越广泛地应用于药物成瘾研究,将有助于了解药物依赖过程中蛋白质的变化及其参与的生物过程和相关通路,理解药物成瘾的分子机制。     

宏蛋白质组学技术在废水生物处理工艺研究领域中的应用

随着后基因组时代的到来,宏蛋白质组学逐渐兴起并在生命科学基础领域和临床医药领域成功运用,宏蛋白质组学技术现已成为各研究领域炙手可热的方法之一.宏蛋白质组学技术在废水生物处理研究领域中的应用刚起步,但已展示其强大功能.本文主要综述近年来国内外宏蛋白质组学在废水生物处理研究领域的研究进展,回顾及总结了宏蛋白质组学的研究策略及应用,如鉴定功能性蛋白质/酶、揭示污染物的微生物降解途径、推断废水生物处理系统的关键代谢途径、及探讨不同污泥微生物群落微生态变化等.
     

蛋白质组学技术在植物类囊体膜蛋白研究中的应用

类囊体作为植物光合作用光反应的重要场所,在植物亚细胞蛋白质组学研究中倍受关注.介绍了植物蛋白质组学相关技术,包括双向凝胶电泳(2DE)、高效液相色谱(HPLC)、高效毛细管电泳(HPCE)、质谱(MS)和蛋白质组学数据库在植物类囊体膜蛋白研究中的应用.同时对类囊体膜蛋白质组学的研究趋势进行了探讨.     

组学技术及其在食品科学中应用的研究进展

后基因组时代的主要研究任务即是组学(转录组学、蛋白质组学及代谢组学)研究,其发展迅速,有望成为解决生命科学领域诸如食品品质与安全等科学问题的有力工具.组学研究为食品科学相关研究提供了新的思路和技术,在食品加工、贮藏、营养素检测、食品安全以及食品鉴伪等领域中已有广泛的应用.综述转录组学、蛋白质组学及代谢组学研究的核心技术,以及组学技术在食品科学研究中的研究进展,并对其应用前景进行展望.     

蛋白质组学技术及其在糖尿病研究中的应用

蛋白质组学是以基因编码蛋白质作为研究对象,并依赖高通量、高自动化的技术对其进行大规模分析的一门学科.其研究方法及手段,已用于糖尿病研究领域.对蛋白质组样品提取技术、蛋白质组分离和分析技术(双向电泳、色谱、质谱分析)、生物信息学以及蛋白质组学技术在研究1型糖尿病(T1DM)、2型糖尿病(T2DM)、胰岛素抵抗等的发病机制以及抗糖尿病药物的开发中的应用等进行了综述.     

大鳞大马哈鱼垂体生长激素的分离、纯化与鉴定

应用ConA-Sepharose 4B亲和层析、凝胶过滤及离子交换层析等技术从大鳞大马哈鱼(Oncorhynchus tshawytscha)垂体中分离纯化了具有生物活性的生长激素(sGH)。用放射受体测定法(RRA)检测sGH组分的生物活性,结果表明纯化的8GH制品具有与兔肝细胞GH受体结合的生物活性。用放射免疫测定法(RIA)和酶联免疫吸附测定法(ELISA)分别检测了另两种垂体激素-催乳激素(PRL)和促性腺激素(GTH)在纯化的sGH制品中的残留量均在0.5％以下。用SDS-聚丙烯酰胺凝胶电泳SDS-PAGE评价sGH制品的电泳纯度并测定了其分子量为22000左右。等电聚焦电泳表明该种鱼GH由等电点分别为6.3和6.6的两种形式的分子组成。     

Titanium dioxide as chemo-affinity chromatographic sorbent of biomolecular compounds--applications in acidic modification-specific proteomics

Characterization of the biomolecules involved in molecular processes occurring in biological systems such as the human cell remains central to biology, biotechnology, and medicine. One of the preferred methods of selectively purifying specific classes of biomolecules from complex biological matrices for further characterization is affinity chromatography, which relies on the specific interaction between an analyte in solution and a solid adsorbent. Titanium dioxide-based affinity chromatography has proven to be a versatile tool in enrichment of various compounds such as phosphorylated biomolecules due to its unique ion and ligand exchange properties and high stability towards pH and temperature. Recently, titanium dioxide chromatography was introduced in proteomics as a highly specific method for enriching phosphorylated peptides - a method, which has been widely adapted by the field of phosphoproteomics. Additional studies have shown the potential of this sorbent in purification of other acidic post-translational modified peptides, such as sialylated glycopeptides, thereby targeting the sialiome, defined as the content of sialic acid containing glycoproteins of a given cell, body fluid or tissue. The development of TiO(2)-based chromatographic strategies for separation of various biomolecules from its introduction for small molecules more than 20 years ago until recent proteomics applications today will be reviewed here.     

Synthesis of marimastat and a marimastat conjugate for affinity chromatography and surface plasmon resonance studies

Immobilized small-molecule inhibitors are suited for enrichment of biomolecules by affinity chromatography, as it is shown for metalloproteinases and an immobilizable derivative of the hydroxamate-type inhibitor marimastat. A new asymmetric synthesis of marimastat is presented that allows for site-specific attachment to a solid surface, e.g., a chromatography matrix or a surface plasmon resonance sensor chip. The latter technique is shown to be a valuable tool for the optimization of binding and elution conditons of biomolecules in affinity chromatography.     

Advances and applications of de novo designed affinity ligands in proteomics

Affinity chromatography represents a promising technique for decoding the proteomics universe. While conventional affinity purification is being used in conjunction with two-dimensional electrophoresis (2D-PAGE) and mass spectrometry (MS) for the study of proteomes and subproteomes, scientists are still confronted with the need for specific and tailor-made affinity ligands to target desired groups and families of proteins. Evidence has shown that, in many situations, synthetic affinity ligands can circumvent inconveniences associated with the utilisation of biological ligands for the chromatography-based purification of biomolecules. This review will highlight the potential applications of affinity chromatography and synthetic de novo designed ligands as separation tools for proteomics.     

Affinity chromatography supports: a look at performance requirements.

Because of its high selectivity, affinity chromatography is a preferred tool in the downstream processing of high-value proteins and peptides of therapeutic interest. This review examines the affinity supports currently available, and investigates the performance characteristics and properties required of the support matrices for improved affinity-based supports for large-scale purification of biomolecules. Parameters for optimizing an affinity chromatographic process, and the advantages of affinity-based separation for scaled-up systems are highlighted.     

Recent advances in bioprocessing application of membrane chromatography

Compared to traditional chromatography using resins in packed-bed columns, membrane chromatography is a relatively new and immature bioseparation technology based on the integration of membrane filtration and liquid chromatography into a single-stage operation. Over the past decades, advances in membrane chemistry have yielded novel membrane devices with high binding capacities and improved mass transfer properties, significantly increasing the bioprocessing efficiency for purification of biomolecules. Due to the disposable nature, low buffer consumption, and reduced equipment costs, membrane chromatography can significantly reduce downstream bioprocessing costs. In this review, we discuss technological merits and disadvantages associated with membrane chromatography as well as recent bioseparation applications with a particular attention on purification of large biomolecules.     

Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA

The diversity of biomolecules present in plasmid DNA (pDNA)-containing extracts and the structural and chemical similarities between pDNA and impurities are some of the main challenges of improving or establishing novel purification procedures. In view of the unequalled specificity of affinity purification, this technique has recently begun to be applied in downstream processing of plasmids. This paper discusses the progress and importance of affinity chromatography (AC) for the purification of pDNA-based therapeutic products. Several affinity approaches have already been successfully developed for a variety of applications, and we will focus here on highlighting their possible contributions to the pDNA purification challenge. Diverse affinity applications and their advantages and disadvantages are discussed, as well as the most significant results and improvements in the challenging task of purifying plasmids.     

Recent developments in downstream processing based on affinity interactions

Novel purification processes have been developed, based on the interaction between complementary biomolecules, to circumvent the difficulties encountered by conventional affinity chromatography. Depending upon the procedure used for isolating the ligand—binder complex, the process can be termed affinity cross-flow filtration, affinity partition or affinity precipitation. This review describes the developments and potentials of such purification techniques.     

Boronic acids for affinity chromatography: spectral methods for determinations of ionization and diol-binding constants

Arylboronic acids attached to solid matrices have proved useful for the diol-specific chromatography of biomolecules and affinity purification of enzymes by exchangeable-ligand chromatography. The latter use has been limited by the intrinsic ionization constant (pKa approximately 9) of the most common commercial products. The synthesis of several arylboronic acids with ionization constants near neutrality are described, and the application of a new general, spectral-difference method for determining acid ionization constants and formation constants with fructose is developed. In particular 4-(N-methyl) carboxamido-benzeneboronic acid was found to have a pKa of 7.86 and a formation constant with D-fructose of 8600. It was stable toward acid or base hydrolysis. We suggest that 4-carboxybenzeneboronic acid might be useful for preparing matrices for enzyme affinity chromatography.     

Affinity as a tool in life science

The use of affinity-based tools has become invaluable as a platform for basic research and in the development of drugs and diagnostics. Applications include affinity chromatography and affinity tag fusions for efficient purification of proteins as well as methods to probe the protein network interactions on a whole-proteome level. A variety of selection systems has been described for in vitro evolution of affinity reagents using combinatorial libraries, which make it possible to create high-affinity reagents to virtually all biomolecules, as exemplified by generation of therapeutic antibodies and new protein scaffold binders. The strategies for high-throughput generation of affinity reagents have also opened up the possibility of generating specific protein probes on a whole-proteome level. Recently, such affinity proteomics have allowed the detailed analysis of human protein expression in a comprehensive manner both in normal and disease tissue using tissue microarrays and confocal microscopy.     

Optimized operating parameters for the displacement separation of biomolecules in immobilized metal ion affinity chromatography

The ideal immobilized metal ion affinity chromatography (IMAC) model was employed to investigate the effect of operating parameters change on the displacement separation of biomolecules. By combining a lower initial mobile phase modifier (MPM) concentration and a higher final MPM concentration, the displacement chromatographic separation produced both higher concentration of feeds and better throughput in IMAC displacement separating systems.