卷柏总蛋白质提取方法及双向电泳条件的建立

目的:建立复苏植物——卷柏总蛋白质的提取方法,以及可以对其蛋白质组进行阵列分离的双向电泳条件。方法:通过多种条件的组合与优化,建立了以物理和化学2种方法充分裂解植物组织细胞,并采用低温操作、加入PVP等去除植物组织中大量的酚类物质,最后离心去除不溶性杂质的卷柏蛋白质组双向电泳(2-DE)样品制备方法。第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE。结果:通过对样品制备、蛋白定量、第1向和第2向电泳、染色方法等各实验环节进行控制和优化,凝胶经考马斯亮蓝染色后,可分辨蛋白质斑点数约600个。结论:建立了卷柏总蛋白质的提取方法及蛋白质组双向电泳技术,为后续研究卷柏在干旱胁迫下的差异表达奠定了基础。     

蛋白质组研究的技术体系及其进展

随着后基因组时代的到来,蛋白质组研究越来越受到国内外科学工作者的密切关注, 我国国家自然科学基金委员会已把蛋白质组研究列为重大科研项目.概述了蛋白质组研究中的基本技术,包括双向凝胶电泳的样品制备和分离、蛋白质的检测、凝胶图像分析、蛋白质的鉴定以及蛋白质数据库构建等,并就蛋白质鉴定的常用方法如氨基酸组成分析方法、蛋白质末端序列分析、肽质量指纹谱作了详细阐述.直观地列出了蛋白质组研究的技术体系流程图,着重介绍了蛋白质组研究的最新技术及其进展.     

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.     

一种适合针刺活检样品的蛋白质组学分析方法

针刺活检样品是一种重要的临床组织样品,其蕴涵的蛋白质信息,对了解人类疾病极为重要.然而,由于该样品的体积极小,其研究受到很大限制.本文建立和优化了适合针刺活检样品的基于双向电泳的蛋白质组学分析平台：通过直接抽提获得针刺活检组织的蛋白质样品;用24 cm 固定梯度干胶条(pH 3-10NL)等电聚焦及12.5% SDS-PAGE分离获得蛋白质样品;感兴趣的蛋白质点经过胰酶酶解后用MALDI TOF/TOF质谱分析.运用所建立的平台对3例来自3只不同大鼠的肝脏针刺活检样品进行分析,获得了多于2500个蛋白质点的高重复性的二维凝胶银染图谱.应用该方法分析人前列腺针刺活检样品的蛋白质组,同样获得了高质量、高重复性的结果.其中随机选取的包括低丰度点在内的57个蛋白质点,经胰酶酶解后进行MALDI串联质谱分析,均获得了高确定性的鉴定结果.通过建立针刺活检样品的蛋白质组学分析方法,为研究人类疾病的分子机制提供了必要的前提保障.     

厌氧产氢颗粒污泥蛋白质组分析样品的高效制备方法

【背景】厌氧产氢颗粒污泥比絮状产氢污泥具有更高的生物量、沉降性与反应效率,对颗粒污泥进行蛋白质组学研究,有助于揭示其代谢调控的分子机制,从而对厌氧代谢过程进行优化调控。目前关于产氢颗粒污泥蛋白质组分析样品制备方法的研究尚未见文献报道。革兰氏阳性菌Ethanoligenens harbinense YUAN-3是自凝集产氢发酵细菌,在间歇和连续流培养中可形成自聚集的厌氧颗粒,由于其全基因组信息清楚,可作为模式研究材料对制备方法进行评估。【目的】针对厌氧产氢颗粒污泥的蛋白质组学研究,比较不同蛋白质提取方法进行优化。【方法】分别利用液氮研磨、超声破碎、匀浆破碎对产氢颗粒污泥破碎,比较这3种方法对总蛋白提取量的影响;通过双向电泳比较三氯乙酸(Trichloroacetic acid,TCA)-丙酮沉淀法与苯酚抽提法对总蛋白提取效果的影响;对总蛋白样品分别进行同位素标记相对和绝对定量标记(Isobarictagsforrelativeandabsolutequantification,i TRAQ)、串联质谱标签(Tandemmasstag,TMT)标记以及质谱鉴定。【结果】液氮研磨、超声破碎、匀浆破碎3种破碎方法下总蛋白的提取量分别是对照样品的2.0、3.9与5.2倍。与TCA-丙酮沉淀法相比,苯酚抽提法总蛋白样品在双向电泳图谱上的蛋白质点明显增多,分布均匀,同时其在碱性蛋白端与小分子量蛋白端的蛋白质点也明显增多。质谱分析发现,iTRAQ标记样品与TMT标记样品中分别鉴定到1797个与1644个蛋白,在分子量、等电点、亚细胞定位的各个分布范围内,这些蛋白良好地覆盖了E.harbinenseYUAN-3中各个类型的蛋白。【结论】匀浆破碎与苯酚抽提法联用的总蛋白制备方法更适用于厌氧产氢颗粒污泥,该方法有利于后续的蛋白质双向电泳和定量蛋白质组质谱分析,可作为产氢颗粒污泥以及革兰氏阳性菌总蛋白制备的方法参考。     

基于二辛基化标记的蛋白质组N末端肽富集方法

蛋白质末端的研究不仅对于蛋白质的结构和功能注释十分必要,还能够为蛋白酶作用机理的揭示提供重要信息.针对传统蛋白质组N末端肽富集需要采用大量去除材料导致N末端肽回收率低,以及步骤繁琐、样品损失等问题,本文发展了一种基于二辛基化标记的蛋白质组N末端肽反向富集方法,可以在单次反相色谱分离中实现非N末端肽的去除.以酵母蛋白质酶解产物为样品对方法进行考察,结果表明,该方法具备较高的二辛基化标记效率(93%),且非N末端肽经二辛基化标记后能够显著偏移至强保留区.将其应用于酵母蛋白质N末端组的分析,共鉴定到237条蛋白原始N末端肽和133 neo-N末端肽,较富集前分别提高了2.1和3.3倍.此外,将该方法结合多种酶切方式,使N末端肽和neo-N末端肽的鉴定数目进一步提高37%和60%,促进了蛋白质N末端组鉴定覆盖度的提高.     

蛋白质组研究中的分析技术

蛋白质组分析技术已在生物科学各领域被广泛应用,蛋白质组的研究已成为目前国际上的前沿和研究热点。概述了蛋白质组研究中的常用分析技术,包括样品制备、双向凝胶电泳、凝胶图像分析、质谱鉴定及数据库检索等。直观地列出了蛋白质组研究的技术体系流程图。各种分析技术的连用和分析过程的自动化将是蛋白质组研究技术的发展方向。     

iTRAQ多重化学标记串联质谱技术在比较蛋白质组学中的应用

研究不同生理和病理条件下细胞内蛋白质的含量和状态变化是比较蛋白质组学的核心内容。要揭示上述动态过程往往需要进行多个样品的同步比较分析。近年来,在体内和体外同位素标记基础上,用多维液相色谱分离多肽,进而用串联质谱进行相对定量的分析方法已成为高通量比较蛋白质组研究的主要手段之一。该文就目前唯一一种可以进行四重蛋白质样品同步比较的iTRAQ标记-串联质谱分析技术进行综述。     

蛋白质芯片技术进展

人类基因组测序工作的完成 ,引起人们对蛋白质组研究的热忱。蛋白质作为生命活动的执行者 ,种类繁多 ,结构复杂 ,并且其活性与空间结构密切相关 ,需要更为先进的技术去研究和探索。近来出现的蛋白质芯片以并行、高通量检测、分析和处理蛋白质样品 ,发展迅速 ,应用前景广泛。介绍蛋白质芯片的种类、蛋白质固定的表面化学以及不同的检测方法 ,简述蛋白质芯片在不同领域的应用 ,并讨论蛋白质芯片目前存在的问题。     

蛋白质组分析技术进展

蛋白质组是指某一物种、个体、器官、组织、细胞乃至体液在精确控制其环境条件之下,特定时刻的全部蛋白质表达图谱。继基因组之后,综的研究即将成为分子生物学的研究热点,蛋白质组研究中常用分离分析技术包括样品制备,双向凝胶电泳,毛细管电泳,色谱技术和质谱技术。双向凝胶电泳是在较短时间内分离大量蛋白质组分,提供足够分离空间的比较成熟的方法。各种分离技术的连用和分析过程的自动化将是蛋白质组研究技术的发展方向。     

Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using isoelectric focusing and SDS-PAGE in the first and second dimensions, respectively, is an established means of simultaneously separating over 1000 proteins and two new types have recently been developed. These procedures have significant shortcomings such as low load ability and poor separation of hydrophobic, acidic and alkaline proteins. We therefore modified the protocols to analyze the Bacillus subtilis membrane proteome. The 2D-PAGE techniques effectively separated membrane proteins having one and two transmembrane segments but not those with more than four. Compared with new LC/MS/MS procedures that are independent of electrophoretic separation, 2D-PAGE can globally analyze and quantify proteins at various stages of the cell cycle when labeled with isotopes such as 35S-methionine or the stable isotope, 15N.     

What place for polyacrylamide in proteomics?

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to deliver high quality protein resolution and dynamic range for the proteomics researcher. To remain as the preferred method for protein separation and characterization, several key steps need to be implemented to ensure quality sample preparation and speed of analysis. Here, we describe the progress made towards establishing 2D-PAGE as the optimal separation tool for proteomics research.     

State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomics technologies. Based on two distinct procedures, it combines isoelectric focusing (IEF), which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. At present, 2D-PAGE is capable of simultaneously detecting and quantifying up to several thousand protein spots in the same gel image. Here we provide comprehensive step-by-step instructions for the application of a standardized 2D-PAGE protocol to a sample of human plasma or cerebrospinal fluid (CSF). The method can be easily adapted to any type of sample. This four-day protocol provides detailed information on how to apply complex biological fluids to an immobilized dry strip gel, cast home-made gradient acrylamide gels, run the gels, and perform standard staining methods. A troubleshooting guide is also included.     

Acoustic technology for high-performance disruption and extraction of plant proteins

Acoustic technology shows the capability of protein pellet homogenization from different tissue samples of soybean and rice in a manner comparable to the ordinary mortar/pestle method and far better than the vortex/ultrasonic method with respect to the resolution of the protein pattern through two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). With acoustic technology, noncontact tissue disruption and protein pellet homogenization can be carried out in a computer-controlled manner, which ultimately increases the efficiency of the process for a large number of samples. A lysis buffer termed the T-buffer containing TBP, thiourea, and CHAPS yields an excellent result for the 2D-PAGE separation of soybean plasma membrane proteins followed by the 2D-PAGE separation of crude protein of soybean and rice tissues. For this technology, the T-buffer is preferred because protein quantification is possible by eliminating the interfering compound 2-mercaptoethanol and because of the high reproducibility of 2D-PAGE separation.     

Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain

Understanding the function of membrane proteins is of fundamental importance due to their crucial roles in many cellular processes and their direct association with human disorders. However, their analysis poses a special challenge, largely due to their highly amphipathic nature. Until recently, analyses of proteomic samples mainly were performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), due to the unprecedented separation power of the technique. However, in conventional 2D-PAGE membrane proteins are generally underrepresented due to their tendency to precipitate during isoelectric focusing and their inefficient transfer from the first to the second dimension. As a consequence, several other separation techniques, primarily based on liquid chromatography (LC), have been employed for analysis of this group of proteins. In the present study, different LC-based methods were compared for the analysis of crude protein extracts. One- and two-dimensional high-performance liquid chromatographic (1D- and 2D-HPLC) separations of brain protein tryptic digests with a predicted concentration range of up to 5 orders of magnitude were found to be insufficient, thus making a preceding fractionation step necessary. An additional protein separation step was introduced and a 3D-PAGE-HPLC analysis was performed. The results of these experiments are compared with results of 2D-PAGE/matrix-assisted laser desorption ionization mass spectrometric (MALDI MS) analyses of the same samples. Features, challenges, advantages, and disadvantages of the respective systems are discussed. The brain (mouse and human) was chosen as the analyzed tissue as it is of high interest in medical and pharmaceutical research into neurological diseases such as multiple sclerosis, stroke, Alzheimer's disease, and Parkinson's disease. The study is part of our ongoing research aimed at identifying new biomarkers for neurodegenerative diseases.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

Size-exclusion chromatography in multidimensional separation schemes for proteome analysis

Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), well-established techniques relying upon similar criteria for separation.Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed.     

Decoding 2D-PAGE complex maps: relevance to proteomics

This review describes two mathematical approaches useful for decoding the complex signal of 2D-PAGE maps of protein mixtures. These methods are helpful for interpreting the large amount of data of each 2D-PAGE map by extracting all the analytical information hidden therein by spot overlapping. Here the basic theory and application to 2D-PAGE maps are reviewed: the means for extracting information from the experimental data and their relevance to proteomics are discussed. One method is based on the quantitative theory of statistical model of peak overlapping (SMO) using the spot experimental data (intensity and spatial coordinates). The second method is based on the study of the 2D-autocovariance function (2D-ACVF) computed on the experimental digitised map. They are two independent methods that are able to extract equal and complementary information from the 2D-PAGE map. Both methods permit to obtain fundamental information on the sample complexity and the separation performance and to single out ordered patterns present in spot positions: the availability of two independent procedures to compute the same separation parameters is a powerful tool to estimate the reliability of the obtained results. The SMO procedure is an unique tool to quantitatively estimate the degree of spot overlapping present in the map, while the 2D-ACVF method is particularly powerful in simply singling out the presence of order in the spot position from the complexity of the whole 2D map, i.e., spot trains. The procedures were validated by extensive numerical computation on computer-generated maps describing experimental 2D-PAGE gels of protein mixtures. Their applicability to real samples was tested on reference maps obtained from literature sources. The review describes the most relevant information for proteomics: sample complexity, separation performance, overlapping extent, identification of spot trains related to post-translational modifications (PTMs).     

Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics

Proteomic analyses of different subcellular compartments, so-called organellar proteomics, facilitate the understanding of cellular functions on a molecular level. In this work, various orthogonal multidimensional separation techniques both on the protein and on the peptide level are compared with regard to the number of identified proteins as well as the classes of proteins accessible by the respective methodology. The most complete overview was achieved by a combination of such orthogonal techniques as shown by the analysis of the yeast mitochondrial proteome. A total of 851 different proteins (PROMITO dataset) were identified by use of multidimensional LC-MS/MS, 1D-SDS-PAGE combined with nano-LC-MS/MS and 2D-PAGE with subsequent MALDI-mass fingerprinting. Our PROMITO approach identified the 749 proteins, which were found in the largest previous study on the yeast mitochondrial proteome, and additionally 102 proteins including 42 open reading frames with unknown function, providing the basis for a more detailed elucidation of mitochondrial processes. Comparison of the different approaches emphasizes a bias of 2D-PAGE against proteins with very high isoelectric points as well as large and hydrophobic proteins, which can be accessed more appropriately by the other methods. While 2D-PAGE has advantages in the possible separation of protein isoforms and quantitative differential profiling, 1D-SDS-PAGE with nano-LC-MS/MS and multidimensional LC-MS/MS are better suited for efficient protein identification as they are less biased against distinct classes of proteins. Thus, comprehensive proteome analyses can only be realized by a combination of such orthogonal approaches, leading to the largest dataset available for the mitochondrial proteome of yeast.