Application of residue distribution along the sequence for discriminating outer membrane proteins

Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important problem both for detecting outer membrane proteins from genomic sequences and for the successful prediction of their secondary and tertiary structures. In this work, we have systematically analyzed the distribution of amino acid residues in the sequences of globular and outer membrane proteins. We observed that the occurrence of two neighboring aliphatic and polar residues is significantly higher in outer membrane proteins than in globular proteins. From the information about the dipeptide composition we have devised a statistical method for discriminating outer membrane proteins from other globular and membrane proteins. Our approach correctly picked up the outer membrane proteins with an accuracy of 95% for the training set of 337 proteins. On the other hand, our method has correctly excluded the globular proteins at an accuracy of 79% in a non-redundant dataset of 674 proteins. Furthermore, the present method is able to correctly exclude alpha-helical membrane proteins up to an accuracy of 87%. These accuracy levels are comparable to other methods in the literature. The influence of protein size and structural class for discrimination is discussed.     

肺鳞癌和小细胞肺癌组织比较蛋白质组学研究

通过16例人肺鳞癌和小细胞肺癌组织中表达蛋白的二维电泳分离和质谱分析,经数据库检索鉴定了53个蛋白,其中24个蛋白与肺癌发病机制相关,4个蛋白在其它癌症中有报道.表达呈现差异的蛋白点有44个,其中34个在表达量上有差异,10个蛋白在鳞癌和小细胞癌间表现为有和无的关系.蛋白功能分析提示人肺鳞癌与小细胞癌的蛋白质组表达存在差异,分析这些差异蛋白有利于肺癌分型及其生物标志物研究.     

4-(2-苄氧基乙氧基羰基)氧杂环丁-2-酮的合成及表征

通过16例人肺鳞癌和小细胞肺癌组织中表达蛋白的二维电泳分离和质谱分析,经数据库检索鉴定了53个蛋白,其中24个蛋白与肺癌发病机制相关,4个蛋白在其它癌症中有报道,表达呈现差异的蛋白点有44个,其中34个在表达量上有差异,lO个蛋白在鳞癌和小细胞癌间表现为有和无的关系,蛋白功能分析提示人肺鳞癌与小细胞癌的蛋白质组表达存在差异,分析这些差异蛋白有利于肺癌分型及其生物标志物研究。     

泉生热袍菌结构基因组的选靶研究

对泉生热袍菌进行了结构基因组的选靶研究，从泉生热袍菌的蛋白组中挑选了20个蛋白质作为第一批进行结构测定的目标，以发现新的蛋白质折叠模式. 选靶研究主要使用了BLAST搜索， PSI-BLAST搜索和ProtoNet数据库搜索等方法. 另外，还用PredictProtein程序对选中的蛋白质进行了二级结构和外形预测. 选中的20个蛋白质中有8个被克隆、表达和纯化，其中2个得到了单晶并收集了X衍射数据. 实验结果和最近一些文献报道的结果表明，挑选的一些蛋白质具有新的折叠模式，表明了这种选靶策略的有效性.     

Defining the plant disulfide proteome

There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.     

Designer peptide surfactants stabilize diverse functional membrane proteins

Multi-spanning integral membrane proteins, including G-protein coupled receptors (GPCR), ion channels, and ion transporters, comprise a major class of drug targets. However, despite their vital importance, most molecular structures of membrane proteins remain elusive. This is largely due to lack of effective materials and methods to stabilize their functional conformation for sufficient time. Thus finding optimal surfactants and developing new approaches to study fundamental properties of unstable membrane proteins is urgently needed. In this tutorial review we summarize designer peptides with surfactant properties and their usefulness to stabilize membrane proteins. These peptide surfactants present new opportunities for the stabilization and characterization of diverse membrane proteins. Previous studies on the interaction between surfactant peptides and membrane proteins revealed strategies to design new peptides tailor-made for the stabilization of specific proteins. We review examples of solubilization, purification, long-term stabilization of membrane proteins, and the design principles of peptide sequences. We discuss future trends for exploiting spatial features, thermodynamic parameters, and self-assembling properties to create peptide surfactant structures to facilitate the characterization of diverse membrane proteins.     

液相等电聚焦结合双向凝胶电泳分离碱性蛋白

在蛋白组学研究中, 经典的双向凝胶电泳法(2-DE)对碱性蛋白及低丰度蛋白的分离存在技术障碍, 但预分离技术的应用可弥补其缺陷. 液相等电聚焦可有效地分离富集复杂蛋白样品. 碱性胶条用于2-DE可极大地提高蛋白上样量和凝胶分辨率. 将上述两种技术相结合用于碱性蛋白质和低丰度蛋白质的分离鉴定, 可使碱端区域双向凝胶图谱质量显著提高, 蛋白点更清晰且点数增多, 质谱鉴定确信度提高, 碱性蛋白和低丰度蛋白质谱鉴定成功率提高, 对于蛋白组学研究具有一定的意义.     

A HMM-based method to predict the transmembrane regions of beta-barrel membrane proteins

A novel method is developed to model and predict the transmembrane regions of beta-barrel membrane proteins. It is based on a Hidden Markov model (HMM) with architecture obeying those proteins' construction principles. The HMM is trained and tested on a non-redundant set of 11 beta-barrel membrane proteins known to date at atomic resolution with a jack-knife procedure. As a result, the method correctly locates 97% of 172 transmembrane beta-strands. Out of the 11 proteins, the barrel size for ten proteins and the overall topology for seven proteins are correctly predicted. Additionally, it successfully assigns the entire topology for two new beta-barrel membrane proteins that have no significant sequence homology to the 11 proteins. Predicted topology for two candidates for beta-barrel structure of the outer mitochondrial membrane is also presented in the paper.     

Enrichment of human brain proteins by heparin chromatography.

Detection of low-abundance proteins is essential for the identification of novel drug targets by differential protein expression studies. We studied the enrichment of human fetal brain proteins by heparin chromatography. Total soluble brain proteins were fractionated on Heparin-Actigel and the fractions collected were analyzed by two-dimensional electrophoresis. The proteins were identified by matrix-assisted laser desorption ionization mass spectrometry. Approximately 300 protein spots were analyzed, representing 70 different polypeptides, 50 of which were bound to the heparin matrix. Eighteen brain proteins were identified for the first time. The proteins enriched by heparin chromatography include both minor and major components of the brain protein extract. The enriched proteins belong to several classes, including proteasome components, dihydropirimidinase-related proteins, T-complex protein 1 components and enzymes with various catalytic activities. The results include a two-dimensional map of the soluble brain proteins and a list of the proteins enriched by heparin chromatography. These may be useful in the design of protein purification protocols and in studies of neurological disorders.     

Membrane proteome in Escherichia coli probed by MS3 mass spectrometry: a preliminary report

Characterization of the membrane proteome is particularly intriguing since a better knowledge in this field might lead to new insights into the function of different membrane systems. Despite the biological relevance of surface proteins however, their characterization still remains a challenging task. Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Hence, surface proteins of Gram-negative bacteria contain proteins that might be good targets for drugs, antimicrobials or detection systems and they may become components of effective vaccines. In this respect, Escherichia coli has been chosen as a model organism for several structural and functional studies aimed at understanding the biophysical and biochemical organization of proteins in Gram-negative cell walls. Here we present first results for the identification of bacterial surface exposed proteins in E. coli K12 based on the use of dansyl chloride labelling coupled with bidimensional tandem mass spectrometry exploiting the advantage of precursor ion/MS3 scan modes. This procedure resulted in a promising, simple, and rapid strategy for the identification of membrane proteins in E. coli as model organism, thus avoiding time-consuming procedures based on two-dimensional liquid chromatography and electrophoresis. The proteins identified could be grouped into five major families: outer membrane (29 proteins), lipoproteins (6 proteins), transmembrane (43 proteins) families.     

Single Molecule Force Spectroscopy on Proteins by AFM——Nanomechanics Meets Molecular Biology

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we combine single molecule force spectroscopy and molecular biology to investigate the nanomechanical properties of these modular proteins. Our experiments reveal the mechanical design of modular proteins, and opens the way for the engineering of elastic proteins with defined and tunable mechanical properties, which can be used in tissue and fiber engineering.     

Cullin-based ubiquitin ligase and its control by NEDD8-conjugating system

Several studies have examined the importance of ubiquitin-like posttranslational modifiers (which consist of an unexpectedly large family). Of these, NEDD8 (also called Rub1, related to ubiquitin 1) with a high homology to ubiquitin is covalently linked to all members of cullin (Cul)-family proteins through an enzymatic cascade analogous to ubiquitylation. Cul-family proteins are scaffold proteins for a wide series of ubiquitin-protein ligase complexes, such as SCFs (Skp1, Cul-1, Roc1, and F-box proteins), which regulate the degradation of broad range of cellular proteins. Unlike ubiquitin, which mostly acts as a degradation signal for the target proteins, NEDD8 acts as an activation signal for Cul-family proteins; i.e., Cul-based ubiquitin-protein ligases. Accordingly, the NEDD8 conjugation pathway regulating Cul-protein function is responsible for a diverse array of biologically important processes, such as the cell cycle progression, signalling cascades and developmental programs. Furthermore, recent studies have revealed that the COP9/Signalosome complex interacts physically and genetically with Cul-family proteins, and catalyzes deconjugation of NEDD8 ligated to Cul-family proteins. This review summarizes recent advances in biochemical and genetic studies on how the NEDD8-modifying system regulates Cul-family proteins and their physiology.     

A rat brain protein expression map including cytosolic and enriched mitochondrial and microsomal fractions

Proteomics is a powerful tool to screen brain protein expression but the methodology is hampered by low abundance of proteins or compartmentalization or overload of high-abundance proteins. It was therefore the aim of the study to determine the expression of brain proteins by using enriched cellular subfractions and pre-electrophoretic chromatographical separation of brain homogenates. We used two-dimensional electrophoresis with subsequent matrix-assisted laser desorption/ionization (MALDI) detection and characterization of brain proteins. Subfractionation into cytosolic, mitochondrial and microsomal compartments was performed by ultracentrifugation. Pre-electrophoretic fractionation of the cytosolic fractions was carried out by ion exchange column chromatography. We detected and identified a large series of 437 proteins in rat brain and have shown proteins specific for the individual subcellular compartments. These proteins included housekeeping, signaling, cytoskeletal, intermediary metabolism, antioxidant proteins on the one and neuron and synaptosomal specific proteins on the other hand. Using fractionations of brain homogenates we were able to improve the power of the method on forming the basis for brain protein expressional studies and providing a reference map as a powerful tool for the neuroscientist.     

Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis

To increase the number of proteins detectable by two-dimensional electrophoresis (2-DE) in plants, we present a new procedure for extracting total proteins from plant tissue. This method avoids any loss of proteins in the course of sample preparation and results in two different fractions, one comprising mainly the cytoplasmatic proteins, the other one containing predominantly structure bond proteins. 2-DE patterns obtained from these two fractions show that the total number of different protein spots detected exceeds the degree of resolution commonly reported for plant proteins threefold.     

Using enrichment index for quality control of secretory protein sample and identification of secretory proteins

Analysis of secretory proteins is an important area in proteomic research. We propose that a good secretory protein sample should be enriched with known secretory proteins, and a secretory protein should be enriched in the secretory protein sample compared with its corresponding soluble cell lysate. Positive identifications of proteins were subjected to quantitation of spectral counts, which reflect relative protein abundance. Enrichment index of the sample (EIS) and the enrichment index for protein (EIP) were obtained by comparing proteins identified in the secretory protein sample and those in the soluble cell lysate sample. The quality of the secretory protein sample can be represented by EIS. EIP was used to identify the secretory proteins. The secretory proteins from mouse dendritic cell sarcoma (DCS) were analyzed by MS. The EISs of two samples were 75.4 and 84.65, respectively. 72 proteins were significantly enriched in secretory protein samples, of which 42 proteins were either annotated in Swiss‐Prot and/or predicted by signal peptides to be secretory. In the remaining 30 proteins, 12 and 15 proteins were positively predicted by SecretomeP and ProP, respectively, and 5 proteins were positive by both methods. Furthermore, 11 proteins were found to be present in exosome in other studies that involved mice dendritic cell lines. We suggest that this assessment method is helpful for systemic research of secretory proteins and biomarker discovery for diseases such as cancer. Copyright © 2008 John Wiley & Sons, Ltd.     

A procedure for the extraction and high resolution two-dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei.

Methods are described for the extraction and preparation of total nuclear proteins for high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post-translational modifications. Unlike other procedures for 2-D PAGE analysis of nuclear proteins the technique allows solubilization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi-labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2-D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2-D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.     

Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography.

Visualization of low-copy-number gene products is essential for the detection of novel drug targets by differential protein expression studies. We investigated the enrichment of low-abundance proteins of Escherichia coli by hydroxyapatite chromatography. The proteins of the various pools collected from a ceramic hydroxyapatite column were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. Approximately 800 spots corresponding to 296 different proteins were identified in the hydroxyapatite eluate. About 130 proteins that had not been detected in the two-dimensional gels of the total extract were identified. Hydroxyapatite chromatography enriched low-abundance but also major components of the E. coli protein extract. In particular, it enriched many low-molecular-mass proteins, such as cold-shock proteins. The proteins bound to the hydroxyapatite matrix belong to several classes, including enzymes with various catalytic activities, heat- and cold-shock proteins and many hypothetical and novel proteins with yet unknown functions. The results include a list of the proteins enriched by hydroxyapatite chromatography and a two-dimensional map of the enriched proteins. They may be useful in the design of protein purification pathways using master purification steps and in the search for novel drug targets.     

快速老化模型小鼠海马蛋白质组学初步研究

应用双向凝胶电泳结合质谱鉴定, 分析比较6月龄和12月龄快速老化模型小鼠(Senescence-accele-rated mouse, SAM)的快速老化亚系SAM-prone/8(SAMP8)及抗快速老化亚系SAM-resistance/1(SAMR1)海马蛋白表达的差异, 从蛋白质水平初步探讨与老化相关的学习记忆功能障碍的发生机制. 结果表明, 与同龄SAMR1比较, 6月龄SAMP8海马中有15个蛋白点表达显著上调, 5个蛋白点表达显著下调; 12月龄SAMP8海马中有12个蛋白点表达显著上调, 2个蛋白点表达显著下调, 2个蛋白点只在SAMP8中有表达. 应用质谱分析结合数据库检索, 共鉴定了22种蛋白质. 6月龄和12月龄SAMP8与SAMR1海马中表达有明显变化的蛋白按功能可分为如下4类: (1) 能量代谢相关蛋白; (2) 线粒体功能相关蛋白; (3) 信号转导相关蛋白; (4) 其它蛋白. 研究结果表明, SAMP8和SAMR1海马蛋白表达存在明显差异, 其中一些蛋白与SAMP8随龄出现的学习记忆功能减退相关, 并可能为研究或发现促智药物作用的新蛋白靶标提供线索.     

Enrichment of low-copy-number gene products by hydrophobic interaction chromatography

Enrichment of proteins in solution is the goal of a purification process and often a scientific challenge. We investigated the capacity of hydrophobic interaction chromatography to enrich proteins, potential candidates for novel drug targets. The soluble protein fraction of Haemophilus influenzae was fractionated over a TSK Phenyl column and the proteins resolved were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Approximately 150 proteins, bound to the column, were identified, 30 for the first time. Most of the proteins enriched by hydrophobic interaction chromatography were represented by major spots, so that an enrichment of low-copy-number gene products was only partially achieved. The proteins enriched by this chromatographic approach belong to various protein classes, including enzymes, ribosomal proteins and proteins with as yet unknown functions. The results include two-dimensional maps and a list of the proteins enriched by hydrophobic interaction chromatography.     

Membrane proteome analysis of the green-sulfur bacterium Chlorobium tepidum

An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including membrane proteins, which are rarely detectable on two-dimensional electrophoresis gels. In this study, different methods were employed for the enrichment of membrane proteins from Chlorobium tepidum prior to analysis with two-dimensional electrophoresis (2-DE). Isolated membranes were solubilized with Triton X-100 and from the supernatant we identified 58 unique proteins. The use of ionic sodium dodecyl sulfate (SDS) for protein solubilization, combined with acetone precipitation, resulted in an improved 2-DE pattern and the total number of the identified proteins was increased to 117. The use of acetone for protein precipitation improved the results by extracting compounds potentially deleterious to the resolution of 2-DE. However, the additional proteins detected by the use of SDS are in the majority more difficult to solubilize than less hydrophobic proteins. Further our attempts for selective extraction of the outer membrane proteins using the acid glycine method allowed the identification of 37 proteins of which 14 were predicted to have a signal sequence indicating their localization in the periplasmic space or in the outer membrane.