Different protein expression of myocardium from Chinese mini-swine model of myocardial infarct

High-resolution two-dimensional gel electrophoresis (2-DE), followed by computer-assisted image analysis was used to screen protein patterns of normal and infarcted myocardial tissues for quantitative and qualitative differences in protein expression. In the gels of pH 5–8 immobilized pH gradient (IPG) strips, 851 protein spots were detected in normal myocardial tissue and 1 032 protein spots were resolved in infarcted myocardial tissue. Thirteen protein spots only expressed in normal myocardial tissue, and 14 protein spots only expressed in infarcted myocardial tissue. Results also showed that 49 protein spots displayed quantitative changes in expression between normal and infarcted myocardial tissue. Eleven protein spots were subjected to mass spectrometry (MS) analysis and seven proteins were identified by peptide mass fingerprinting (PMF). These proteins may be involved in cardiovascular injury, and could play an important role in the treatment of coronary heart disease. __________ Translated from Chemical Journal of Chinese Universities, 2006, 8(27): 1467–1471 [译自: 高等学校化学学报]     

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.     

固定化酶反应器及其在蛋白质组研究中的应用

综述了近年来国内外酶固定化载体的研究进展,侧重于无机材料和有机聚合物材料上的固定化酶方法;此外,也介绍了固定化胰蛋白酶反应器与分离系统联用在蛋白质样品分析中的应用,并展望了固定化酶反应器的研究方向及其在蛋白质组中的应用前景。     

基于共价色谱分离的含半胱氨酸肽段富集策略

多维色谱分离、串联质谱分析技术已在蛋白质组研究中得到广泛应用。然而生物样品的蛋白质以及全酶切肽段具有高度的复杂性,这严重干扰了蛋白质高通量、规模化的分析。通过标签肽段富集进行样品预分离可以降低体系的复杂程度。本文建立了一种基于共价色谱技术选择性分离富集含半胱氨酸肽的方法,从而降低了样品体系的复杂程度。首先以牛血清白蛋白(BSA)的酶切肽段为模型,对富集条件进行了优化和考察,并在此基础上通过5种蛋白质酶切肽段混合物的富集对该方法进行了验证。结果证明此方法的重现性好,富集效率高,富集特异性好,能有效地富集鉴定含半胱氨酸肽段。所建立的方法在复杂体系的蛋白质组研究中具有广泛的应用前景,为复杂样品的蛋白质高通量、自动化、规模化鉴定和定量研究提供了实用技术。     

基于生物质谱技术的磷酰化修饰策略在多肽测序中的应用

该文建立了一种利用磷酰化修饰结合电喷雾质谱(ESI-Q-TOF)测定多肽氨基酸序列的有效方法。利用Atherton-Todd反应,以二丙基亚磷酰酯(DPP)为磷酰化试剂,应用生物质谱技术,对磷酰化修饰后的5种模型肽的磷酰化反应情况进行了系统研究,考察了磷酰化肽的二级质谱特征,并与未经磷酰化反应的肽的二级质谱特征对比。结果表明,经过磷酰化修饰后,肽的二级质谱中的a1离子信号强度明显增加,可以准确鉴定其N端氨基酸;b系列离子信息完整,信号强度增强,使得多肽C ID测序的谱图简单、清晰,有利于肽的氨基酸序列的测定;赖氨酸(K,128.10 u)和谷氨酰胺(Q,128.13 u)两种氨基酸质荷比相近,由于二者磷酰化修饰后的差异性,使其得到准确区分。经过5种已知氨基酸序列的模型肽的磷酰化后结合质谱技术进行氨基酸序列测定验证,结果表明该方法简单、快速、准确,提高了利用质谱技术进行多肽测序的准确度和灵敏度,可为蛋白质组学研究提供有效的技术手段。     

分泌蛋白质组研究进展

分泌蛋白质组是指组织、细胞等分泌的全部蛋白质。分泌蛋白主要分布于体液和细胞质胞浆中,参与许多重要的生命过程。分泌蛋白质组的研究主要涉及分泌蛋白的制备、多维色谱或二维凝胶电泳分离、质谱鉴定及生物信息学分析等。本文主要介绍了分泌蛋白的合成途径、蛋白质组技术在分泌蛋白组研究中的应用、分泌蛋白组研究现状及存在的问题等。     

Application of displacement chromatography for the proteome analysis of a human plasma protein fraction

It was the aim of this study to compare the performance of displacement chromatography with gradient elution chromatography both applied as the cation-exchange separation step for a proteome analysis in a bottom-up approach using multidimensional chromatography for the separation of tryptic peptides prior to their mass spectrometric analysis. The tryptic digest of the human Cohn fraction IV-4 served as a sample. For both chromatography modes commonly used operating parameters were chosen thus ensuring optimal separation results of equal sample amounts for each mode. All resulting fractions were analyzed with an HPLC-chip–LC–MS system. The eluate of the HPLC-chip column was ionized by electrospray ionization (ESI) and analyzed with an ion-trap mass spectrometer. For guaranteeing high confidence concerning the identity of the peptides, the mass spectrometric data were processed by different bioinformatic tools applying stringent criteria. By the displacement approach the total amount of identified proteins (78) was significantly higher than in the gradient mode (58). The results showed that displacement chromatography is a well suited alternative in comparison to gradient elution separation for analysis of proteomes via the bottom-up approach applying multidimensional chromatography, especially in those cases when larger quantities of proteins are available.     

Proteomic analysis of a spring wheat cultivar in response to prolonged cold stress

Cold represents one of the major abiotic factors influencing plant growth and development worldwide. We analysed the long-term responsiveness of an Iranian spring wheat (cv. Kohdasht) to cold from a proteomic point of view, in order to unravel the molecular mechanisms helping a cold-sensitive cultivar to survive exposure to suboptimal temperatures. Plants were grown at 20 or 4°C until entering the reproductive stage and a cross-comparison on the leaf proteomes was performed. Quantitative analyses on protein alterations occurring upon low-temperature exposure showed a reinforcement in ascorbate recycling (dehydroascorbate reductase, ascorbate peroxidase) and protein processing (proteasome subunit, cysteine proteinase), as well as the accumulation of the enzyme devoted to tetrapyrrole resynthesis (glutamate semialdehyde aminomutase). In contrast, among proteins down-regulated after cold stress, we could identify some key Krebs cycle enzymes (isocitrate dehydrogenase, malate dehydrogenase), together with many photosynthesis-related proteins (oxygen-evolving complex proteins, ATP synthase subunits, ferredoxin NADPH oxidoreductase and some Calvin cycle enzymes). Physiological and biochemical parameters (such as shoot apex dissection, chlorophyll, proline and sugar content determination) sustained proteomics findings allowing the present research to contribute to the current knowledge on these long-term responses, which may be crucial to stress adaptation under field conditions.     

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.     

Differential proteome analysis of colon carcinoma cell line SW480 after reconstitution of the tumour suppressor Smad4

The tumour suppressor gene Smad4 is frequently inactivated in gastrointestinal carcinomas. Smad4 plays a pivotal role in transducing signals of the transforming growth factor-β (TGF-β) superfamily of proteins. Inactivation of Smad4 seems to occur late during tumour progression when tumours acquire invasive and metastatic properties. Identification of proteins directly or indirectly regulated by Smad4 would, therefore, ease the future design of new diagnostic and therapeutic strategies for gastrointestinal carcinoma. We have used human colon carcinoma cell line SW480 stably transfected with Smad4 as an in-vitro model system to identify Smad4-regulated proteins by applying two-dimensional gel electrophoresis (2DE) then MALDI-PMF/PFF-MS. We identified a total of 47 protein species with a Smad4-dependent expression. From the functions of the candidate proteins we obtained new insights into Smad4’s participation in processes, for example apoptosis, differentiation, and proliferation.