An electrochemical molecular recognition-based aptasensor for multiple protein detection

This article reports a simple electrochemical approach for the detection of multiple proteins (thrombin and lysozyme) using Dabcyl-labeled aptamer modified metal nanoparticles (DLAPs). DLAPs were immobilized on β-cyclodextrins (β-CDs) modified electrode by means of host–guest self-assembly. During the time of detection, the aptamers' structure will change due to the specific binding with corresponding proteins that forced DLAPs far away from the electrode that had been modified by β-CDs. Thus, the capture of target proteins onto DLAPs was translated via the electrochemical current signal offered by metal nanoparticles. Linearity of the aptasensor for quantitative measurements was demonstrated. Determinations of proteins in human real serum samples were also performed to demonstrate detection in real clinical samples.     

Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks

Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.     

Integration of Jacobson's pellicle method into proteomic strategies for plasma membrane proteins

A modified form of the cationic colloidal silica technique for plasma membrane isolation has been combined with SDS-PAGE, mass spectrometry, and bioinformatics for evaluation as a proteomics strategy with human multiple myeloma cells and human breast cancer cells. On the basis of Western blots, half of the protein isolated is estimated to come from the plasma membrane. Forty-three percent of the 366 proteins identified by mass spectrometry had been previously classified as plasma membrane proteins. Thirty proteins previously categorized as hypothetical membrane proteins are now reported to be expressed.     

Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane.

We have developed a new detection method of deiminated proteins on polyvinylidene difluoride membranes. Citrulline residues in enzymatically deiminated histones were modified by incubating with diacetyl monoxime and antipyrine in a strong acid mixture. The products were injected to rabbits, and the antibodies obtained were affinity-purified using a modified citrulline column. Sample proteins blotted to the membrane were modified in a similar manner and incubated successively with the purified antibody and an alkaline phosphatase-conjugated second antibody. Detection was performed using a chemiluminescent substrate. The method enabled detection of 3-10 fmol of citrulline residues dot blotted as deiminated model proteins. It visualized numerous rat pituitary soluble proteins that had been enzymatically deiminated and Western blotted to the membrane. The data suggest usefulness of the method for detecting deiminated proteins regardless of the backbone protein molecules. Search for deiminated proteins on the Western blots of various rat tissue homogenates detected a single band on that of spinal cord, another band on that of uterus, and multiple bands on those of skin and hair root. The bands in the former two tissue homogenates comigrated with glial fibrillary acidic protein and vimentin, respectively.     

Cartilage proteoglycan aggregate is degraded more extensively by cathepsin L than by cathepsin B.

The degradative actions of cathepsins L and B on human articular-cartilage proteoglycan aggregates were examined. Cathepsin L was found to be much more extensive than cathepsin B in degrading proteoglycan aggregates. It released products with size similar to that of single chondroitin sulphate chains, and a series of degraded link-protein fragments in the digestion mixtures. These proteolytically modified link-protein components (Mr 25,000 and 33,000) have similar Mr values to those of fragments observed in adult human cartilage. In contrast, cathepsin B exhibited a much more limited degradation on both proteoglycan subunits and link-protein components. Both cathepsins L and B generate multiple but distinct cleavage sites on human link proteins, and the hydrolysed bonds have been identified in the region between residues 18 and 29. Protein sequencing analysis of these modified link-protein components also provided evidence for the location of a second N-linked glycosylation site at residue 41 in human link proteins, in addition to that previously described at residue 6 on a proportion of the link proteins. Furthermore, it allows us to report the sequence of human link protein up to residue 65.     

Two-dimensional map of membrane proteins from human erythrocytes

Human erythrocyte membrane proteins were analyzed by a modified two-dimensional electrophoresis performed according to O'Farrell. This method was used to construct a two-dimensional map of human erythrocyte membrane proteins. The map plotted in the coordinates "relative molecular mass versus relative electrophoretic mobility during IEF" was used for the characterization of 189 proteins. The position of major membrane proteins in the map was determined on the basis of their Mr, pI as well as literature data. Carboanhydrase was positioned by coelectrophoresis. A comparative analysis of erythrocyte membrane and cytosol preparations by two-dimensional protein mapping revealed that some of erythrocyte proteins have dual localization.     

Properties of thrombin- and elastase-modified human antithrombin III

Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of modification by ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins (Ubls) are conjugated to many target proteins either as monomeric units or as polymeric chains. There are at least 12 members of the ubiquitin family in the human genome and their conjugation dramatically alters the properties of the modified protein. The presence of highly active proteases that specifically deconjugate Ubls often means that, in the cell, the steady state level of modified protein is low. Detection of protein species modified by Ubls can therefore represent a significant challenge. Here, we describe methods that have been developed to allow detection of Ubl modified proteins both in vivo and in vitro.     

Production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview

During the past two decades, antibodies, antibody derivatives and vaccines have been developed for therapeutic and diagnostic applications in human and veterinary medicine. Numerous species of dicot and monocot plants have been genetically modified to produce antibodies or vaccines, and a number of diverse transformation methods and strategies to enhance the accumulation of the pharmaceutical proteins are now available. Veterinary applications are the specific focus of this article, in particular for pathogenic viruses, bacteria and eukaryotic parasites. We focus on the advantages and remaining challenges of plant-based therapeutic proteins for veterinary applications with emphasis on expression platforms, technologies and economic considerations.     

An affinity-based method for the purification of fluorescently-labeled biomolecules

Due to the difficulty of separating mixtures of labeled and unlabeled biomolecules, a general new method for the affinity purification of modified proteins has been developed. A Sepharose-based solid support bearing beta-cyclodextrin groups was used to capture chromophore-modified proteins selectively, while unmodified proteins remained in solution. After isolation of the resin, the modified proteins were released by treating the sample with a competitive cyclodextrin binder, such as adamantane carboxylic acid. This procedure was demonstrated for several dyes displaying a wide range of spectral characteristics and diverse chemical structures. Preliminary studies have shown that this method can also be used to enrich modified peptide fragments present in proteolytic digests. This technique is anticipated to accelerate the development of new protein modification reactions and could provide a useful tool for proteomics applications.     

A simple and effective method for protein subcellular localization using <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of onion epidermal cells

A modified Agrobacterium-mediated transformation protocol has been successfully used for transient expression of the intrinsically fluorescent proteins and their fusion proteins in onion epidermis. The mean of the transformed cells rate per peel is about 10.5±0.9%, while that of the particle bombardment method is at the range 2.0±0.4%. To compare with the prevailing method of micro-projectile bombardment, the modified Agrobacterium-mediated transformation may provide with higher efficiency and even more simplified manipulability on a lower budget.     

A Method for the Parallel and Sequential Generation of Single-Domain Antibodies for the Proteomic Analysis of Human Blood Plasma

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.     

重组人β趋化因子RANTES基因融合表达产物活性分析

为研制基于病毒受体的抗艾滋病毒感染基因药物 ,将重组人 β趋化因子RANTES基因导入大肠杆菌中表达谷胱甘肽S 转移酶 (GST) RANTES融合蛋白 ,并回收携带末端延伸肽 (TEP)的RANTES修饰蛋白 .荧光免疫化学分析表明 ,2种非天然RANTES蛋白均显示对人外周血淋巴细胞的结合活性 .暗示在细胞表面可能已发生RANTES与CCR5之间的相互作用 .2种修饰RANTES蛋白都能使人外周血细胞的过氧化物酶活性显著升高 ,提示这 2个人造蛋白可能仍存在对淋巴细胞的趋化性诱导 .     

Improved resolution with one-dimensional polyacrylamide gel electrophoresis: myofibrillar proteins from typed single fibers of human muscle

Standard procedures for one-dimensional discontinuous sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining were modified to give more effective separation and an improved resolution of human skeletal muscle proteins. In this system, an electrophoresis buffer composed of 100 mM L-isoleucine, 25 mM Tris base, and 0.1% SDS was used. The separating gel consisted of 16% acrylamide with N,N'-methylenebisacrylamide as a crosslinker (1:23), 0.4% SDS, 1.5 M Tris-HCl, pH 8.8. By the present procedure, the slow and the fast forms of myosin light chains (LCs, LCf) and other contractile proteins from human muscle could be better separated. The silver stain is based on a combination of methods previously described. The modified method requires a small fragment of a single fiber to observe as few as 10 ng of myofibrillar muscle proteins. The described simplifications made it possible to assay and compare up to 40 single fibers in the same electrophoretic run. Improved separation of other proteins migrating at basic pH could be achieved by a similar approach.     

Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.     

An efficient algorithm for large-scale detection of protein families

Detection of protein families in large databases is one of the principal research objectives in structural and functional genomics. Protein family classification can significantly contribute to the delineation of functional diversity of homologous proteins, the prediction of function based on domain architecture or the presence of sequence motifs as well as comparative genomics, providing valuable evolutionary insights. We present a novel approach called TRIBE-MCL for rapid and accurate clustering of protein sequences into families. The method relies on the Markov cluster (MCL) algorithm for the assignment of proteins into families based on precomputed sequence similarity information. This novel approach does not suffer from the problems that normally hinder other protein sequence clustering algorithms, such as the presence of multi-domain proteins, promiscuous domains and fragmented proteins. The method has been rigorously tested and validated on a number of very large databases, including SwissProt, InterPro, SCOP and the draft human genome. Our results indicate that the method is ideally suited to the rapid and accurate detection of protein families on a large scale. The method has been used to detect and categorise protein families within the draft human genome and the resulting families have been used to annotate a large proportion of human proteins.     

Detection and quantification of citrullinated chemokines

Background

Posttranslational deimination or citrullination by peptidylarginine deiminases (PAD) regulates the biological function of proteins and may be involved in the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This posttranslational modification of arginine was recently discovered on inflammatory chemokines including CXCL8 and CXCL10, and significantly reduced their biological activity. To evaluate the importance of these modified chemokines in patients, methods for the detection and quantification of citrullinated chemokines are needed. Since citrullination only results in an increase of the protein mass with one mass unit and the loss of one positive charge, selective biochemical detection is difficult. Therefore, we developed an antibody-based method to specifically detect and quantify citrullination on a protein of interest.

Methodology/Principal Findings

First, the citrullinated proteins were chemically modified with antipyrine and 2,3-butanedione at low pH. Such selectively modified citrullines were subsequently detected and quantified by specific antibodies raised against a modified citrulline-containing peptide. The specificity of this two-step procedure was validated for citrullinated CXCL8 ([Cit⁵]CXCL8). Specific detection of [Cit⁵]CXCL8 concentrations between 1 and 50 ng/ml was possible, also in complex samples containing an excess of contaminating proteins. This novel detection method was used to evaluate the effect of lipopolysaccharide (LPS) on the citrullination of inflammatory chemokines induced in peripheral blood mononuclear cells (PBMCs) and granulocytes. LPS had no significant effect on the induction of CXCL8 citrullination in human PBMCs and granulocytes. However, granulocytes, known to contain PAD, were essential for the production of significant amounts of [Cit⁵]CXCL8.

Conclusion/Significance

The newly developed antibody-based method to specifically detect and quantify chemically modified citrullinated proteins is proven to be effective. This study furthermore demonstrates that granulocytes were essential to obtain significant levels of [Cit⁵]CXCL8. For human PBMCs and granulocytes stimulation with LPS did not affect the citrullination of CXCL8.     

Effect of cobalt on <Emphasis Type="Italic">Escherichia coli</Emphasis> metabolism and metalloporphyrin formation

Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe–S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS.     

Quantitative determination of SH groups in low- and high-molecular-weight compounds by an electron spin resonance method

To quantitatively determine SH groups in high- and low-molecular-weight compounds, a disulfide biradical (RS-SR), where R is imidazoline residue, has been used. The biradical is shown to participate in a thiol-disulfide exchange reaction with compounds containing SH groups. In this case the ESR spectra of the biradical RS-SR and the resulting monoradical R-SH are different. The reaction of the biradical with cysteine, glutathione, and human serum albumin has been studied using the ESR method and the rate constants kf of this reaction have been calculated. Studies of the pH dependence of kf indicate that the thiol-disulfide exchange occurs by reaction with mercaptidione. Protein human serum albumin and hemoglobin have been modified by RS-SR. It has been shown that the treatment of modified proteins with reduced glutathione leads to removal of the radical from the protein; such modifications are thus reversible. The method proposed has been used to quantitatively determine the SH groups of cysteine and glutathione in mouse and rat blood. The method is shown to coincide within experimental error with the determination of glutathione and cysteine by titration with p-chloromercuribenzoate or reaction with Ellman's reagent. This method allows detection of 10(-6)-10(-7) M SH compounds even in colored and highly absorbing samples. The kinetics of the SH group modification can also be determined, leading to deduction about accessibility of the SH group in protein.     

Human plasma proteome analysis by reversed sequence database search and molecular weight correlation based on a bacterial proteome analysis

In shotgun proteomics, proteins can be fractionated by 1-D gel electrophoresis and digested into peptides, followed by liquid chromatography to separate the peptide mixture. Mass spectrometry generates hundreds of thousands of tandem mass spectra from these fractions, and proteins are identified by database searching. However, the search scores are usually not sufficient to distinguish the correct peptides. In this study, we propose a confident protein identification method for high-throughput analysis of human proteome. To build a filtering protocol in database search, we chose Pseudomonas putida KT2440 as a reference because this bacterial proteome contains fewer modifications and is simpler than the human proteome. First, the P. putida KT2440 proteome was filtered by reversed sequence database search and correlated by the molecular weight in 1-D-gel band positions. The characterization protocol was then applied to determine the criteria for clustering of the human plasma proteome into three different groups. This protein filtering method, based on bacterial proteome data analysis, represents a rapid way to generate higher confidence protein list of the human proteome, which includes some of heavily modified and cleaved proteins.