One step microelectroelution concentration method for efficient coupling of sodium dodecylsulfate gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry for protein analysis

The coupling of the widely used separation technique of conventional sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with the mass accuracy measurement capability of mass Spectrometry (MS) provides a very powerful analytical technique. However, at present, there is no simple, definitive method for coupling the two methods. Typically, separated proteins are extracted from the gel, either as the native protein or as a peptide mixture after in-gel proteolytic digestion, and then analyzed by mass Spectrometry. However, the various extraction techniques described previously have been labor intensive and require a large number of steps. The mass Spectrometry analysis of very low concentrations of in vivo derived proteins requires minimum sample handling and on-line concentration. Therefore, we have developed an efficient microelectroelution technique that is applied in a single step manner and contains an on-line concentration device. Initial results from this system have shown a high efficiency of analyte elution from the gel and a simple, robust technique for the coupling of SDS-containing gels with MALDI-TOF-MS analysis and a capability of analyzing proteins at the subpicomole level.     

Reversed-phase chromatography of proteins and peptides on compressed continuous beds

Summary The polymer beds described are synthesized in aqueous solution directly in the column or batchwise in the form of large clusters of small particles. The conventional, expensive step involving prepreparation of beads in an organic solvent is thus omitted. Beds were synthesized from piperazine diacrylamide, methacrylamide and allyl glycidyl ether. The epoxy-activated beds thus obtained were used for covalent attachment of either nonpolar ligands (e.g. octadecanol) or polar OH-rich substances (e.g. dextran). The non-polar beds were used for reversed-phase chromatography, as were polar ones following coupling with 1,2-epoxyoctadecane. Coating with OH-rich substances serves two purposes: (I) the matrix becomes hydrophilic, decreasing nonspecific interactions (modifiers can be excluded) and hence increases resolution and (II) many—OH groups are available (e.g. for coupling to epoxides), a prerequisite for high ligand density. Resolution of proteins was high even at high flow rates. Depending on the method used for the synthesis of the bed, resolution of proteins either increased with an increase in flow rate or decreased slinghtly. Choice of the correct temperature was very important for high resolution of CNRr-digested peptides.     

Modular Architecture of Protein Binding Units for Designing Properties of Cellulose Nanomaterials

Molecular biomimetic models suggest that proteins in the soft matrix of nanocomposites have a multimodular architecture. Engineered proteins were used together with nanofibrillated cellulose (NFC) to show how this type of architecture leads to function. The proteins consist of two cellulose‐binding modules (CBM) separated by 12‐, 24‐, or 48‐mer linkers. Engineering the linkers has a considerable effects on the interaction between protein and NFC in both wet colloidal state and a dry film. The protein optionally incorporates a multimerizing hydrophobin (HFB) domain connected by another linker. The modular structure explains effects in the hydrated gel state, as well as the deformation of composite materials through stress distribution and crosslinking. Based on this work, strategies can be suggested for tuning the mechanical properties of materials through the coupling of protein modules and their interlinking architectures.     

Computational prediction of the coupling specificity of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent one of the most important categories of membrane proteins that play important roles in signaling pathways. GPCRs transduce the extracellular stimuli into intracellular second messengers via their coupling to specific class of heterotrimeric GTP-binding proteins (G proteins) and the subsequent regulation of a diverse variety of effectors. Understanding the coupling specificity of GPCRs is critical for further comprehending their function, and is of tremendous clinical significance because GPCRs are the most successful drug targets. This minireview addresses the computational approaches that have been created for the prediction of coupling specificity of GPCRs and highlights the perspective of bioinformatics strategies that may be used to tackle this important task. In addition, some of the important resources of this field are also provided.     

Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection

A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment. Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension. Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest. A peak capacity of more than 10 000 had been achieved. A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest. FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.     

The influence of non-rubber constituents on performance of silica reinforced natural rubber compounds

An in-rubber study of the interaction of silica with proteins present in natural rubber show that the latter compete with the silane coupling agent during the silanisation reaction; the presence of proteins makes the silane less efficient for improving dispersion and filler–polymer coupling, and thus influences the final properties of the rubber negatively. Furthermore, the protein content influences the rheological properties as well as filler–filler and filler–polymer interactions. Stress strain properties also vary with protein content, as do dynamic properties. With high amounts of proteins present in natural rubber, the interactions between proteins and silica are able to disrupt the silica–silica network and improve silica dispersion. High amounts of proteins reduce the thermal sensitivity of the filler–polymer network formation. The effect of proteins is most pronounced when no silane is used, but they are not able to replace a coupling agent.     

Electronic structure contributions to electron-transfer reactivity in iron-sulfur active sites: 3. Kinetics of electron transfer

The kinetics of electron transfer for rubredoxins are examined using density functional methods to determine the electronic structure characteristics that influence and allow for fast electron self-exchange in these electron-transport proteins. Potential energy surfaces for [FeX(4)](2-,1-) models confirm that the inner-sphere reorganization energy is inherently small for tetrathiolates ( approximately 0.1 eV), as evidenced by the only small changes in the equilibrium Fe-S bond distance during redox (Deltar(redox) approximately 0.05 A). It is concluded that electronic relaxation and covalency in the reduced state allow for this small in this case relative to other redox couples, such as the tetrachloride. Using a large computational model to include the protein medium surrounding the [Fe(SCys)(4)](2-,1-) active site in Desulfovibrio vulgaris Rubredoxin, the electronic coupling matrix element for electron self-exchange is defined for direct active-site contact (H0(DA)). Simple Beratan-Onuchic model is used to extend coupling over the complete surface of the protein to provide an understanding of probable electron-transfer pathways. Regions of similar coupling properties are grouped together to define a surface coupling map, which reveals that very efficient self-exchange occurs only within 4 sigma-bonds of the active site. Longer-range electron transfer cannot support the fast rates of electron self-exchange observed experimentally. Pathways directly through the two surface cysteinate ligands dominate, but surface-accessible amides hydrogen-bonded to the cysteinates also contribute significantly to the rate of electron self-exchange.     

Antibodies immobilized on inorganic supports

Antibodies and antigens can be covalently coupled to a variety of carriers, both organic and inorganic. The methods for coupling these proteins may be found scattered throughout the technical literature. This report, although it concentrates on inorganic supports, describes several of the more successful methods used in laboratories today. Each of these methods is described in enough detail for the reader to carry out the coupling method of interest in his or her own laboratory. The coupling methods have been divided into two groups, direct and silane. Under each of these general headings are described the specific methodologies.     

Organic functionalization of luminescent oxide nanoparticles toward their application as biological probes

Luminescent inorganic nanoparticles are now widely studied for their applications as biological probes for in vitro or in vivo experiments. The functionalization of the particles is a key step toward these applications, since it determines the control of the coupling between the particles and the biological species of interest. This paper is devoted to the case of rare earth doped oxide nanoparticles and their functionalization through their surface encapsulation with a functional polysiloxane shell. The first step of the process is the adsorption of silicate ions that will act as a primary layer for the further surface polymerization of the silane, either aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GPTMS). The amino- or epoxy- functions born by the silane allow the versatile coupling of the particles with bio-organic species following the chemistry that is commonly used in biochips. Special attention is paid to the careful characterization of each step of the functionalization process, especially concerning the average number of organic functions that are available for the final coupling of the particles with proteins. The surface density of amino or epoxy functions was found to be 0.4 and 1.9 functions per square nanometer for GPTMS and APTES silanized particles, respectively. An example of application of the amino-functionalized particles is given for the coupling with alpha-bungarotoxins. The average number (up to 8) and the distribution of the number of proteins per particle are given, showing the potentialities of the functionalization process for the labeling of biological species.     

Photopatterned surfaces for site-specific and functional immobilization of proteins

Photosensitive silanes containing nitroveratryl (Nvoc)-caged amine groups and protein repellent tetraethylene glycol units were synthesized and used for modification of silica surfaces. Functional surface layers containing different densities of caged amine groups were prepared and activated by UV-irradiation of the surface. The performance of these layers for functional and site-selective immobilization of proteins was tested. For this purpose, biotin and tris-nitrilotriacetic acid (tris-NTA) were fist coupled to the activated surface, and the interaction of streptavidin and His-tagged proteins with the functionalized surfaces was monitored by real-time label-free detection. After optimizing the coupling protocols, highly selective functionalization of the deprotected amine groups was possible. Furthermore, the degree of functionalization (and therefore the amount of immobilized protein) was controlled by diluting the surface concentration of the amine-functionalized silane with a nonreactive (OMe-terminated) tetraethylene glycol silane. Immobilized proteins were highly functional on these surfaces, as demonstrated by protein-protein interaction assays with the type I interferon receptor. Protein micropatterns were successfully generated after masked irradiation and functionalization of the caged surface following the optimized coupling protocols.     

High-sensitive protein analysis by FESI-CE-MALDI-MS

Capillary zone electrophoresis (CZE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) are two techniques highly suitable for the separation and detection of intact proteins. Herein, based on the use of a recently introduced iontophoretic fraction collection interface for the coupling of CE and MALDI-MS, the potential of the combination of both techniques for the analysis of intact proteins is assessed. To further provide a bioanalytical platform with high-sensitivity capabilities, field-enhanced sample injection is integrated as on online preconcentration strategy upstream from the electrokinetic separation. Under optimized conditions, more than 3200- and 4800-fold improvement, respectively in terms of peak height and peak area, as well as LODs ranging from 5 to 10 nM, has been achieved.     

Synthesis and properties of monofluorinated dimyristoylphosphatidylcholine derivatives: potential fluorinated probes for the study of membrane topology

The synthesis of three monofluorinated dimyristoylphosphatidylcholine derivatives (F-DMPC), with the fluorine atom located on the acyl chain in position 2 of the glycerol (sn-2) is described. The synthetic strategy relies on the coupling of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) and three different fluorinated fatty acids. The latter were obtained from two different and complementary synthetic routes. Preliminary FTIR studies suggest that the presence of the fluorine atom does not significantly perturb the lipid conformational order and phase transition temperature and that these monofluorinated PC derivatives could be used as probes for the study of membrane topology, i.e. the location of drugs, peptides or proteins in membranes.     

空间排阻色谱(SEC)用多孔硅胶载体的研究

我们的工作是制备多孔硅球表面键合带有羟基基团的亲水性SEC刚性载体.研究结果表明,此种SEC载体能适用于聚乙烯醇、明胶、蛋白质、右旋糖酐等多种水溶液体系的SEC分析.     

Preparation of adsorbents for affinity chromatography using TSKgel Tresyl-Toyopearl 650M

The optimum conditions for the coupling of proteins were investigated using TSKgel Tresyl-Toyopearl 650M. They were dependent on the proteins coupled. For example, when soybean trypsin inhibitor was coupled at pH 8 the coupling was completed within 1 h and the subsequent adsorption capacity for trypsin was maximal. Longer coupling times decreased the adsorption capacity due to multi-point attachment. The adsorbents obtained were successfully used for affinity chromatography in a short time.     

Switch in Catalyst State: Single Bifunctional Bi-state Catalyst for Two Different Reactions

Disclosed here is a molecular switch which responds to acid-base stimuli and serves as a bi-state catalyst for two different reactions. The two states of the switch serve as a highly active and poorly active catalyst for two catalytic reactions (namely a hydrogenation and a dehydrogenative coupling) but in a complementary manner. The system was used in an assisted tandem catalysis set-up involving dehydrogenative coupling of an amine and then hydrogenation of the resulting imine product by switching between the respective states of the catalyst.     

One-pot labeling and purification of peptides and proteins with fluorescein maleimide

Fluorescein labeling of peptides and proteins is required for numerous biophysical or biological experiments such as fluorescence microscopy, fluorescence resonance energy transfer (FRET) or fluorescence imaging. The commonly used strategy relied on the coupling of the dye reagent followed by a gel filtration to recover the labeled molecule. Here we report a simplified method for the labeling of peptides and proteins on a cysteine residue and their purification. The method is based on the precipitation of peptides and proteins in acetone, fluorescein maleimide being soluble in this solvent. The excess of dye is fully eliminated after a couple of acetone washes and the precipitated peptide or protein is readily recovered.     

Circular and linear dichroism of proteins

Circular dichroism (CD) is an important technique in the structural characterisation of proteins, and especially for secondary structure determination. The CD of proteins can be calculated from first principles using the so-called matrix method, with an accuracy which is almost quantitative for helical proteins. Thus, for proteins of unknown structure, CD calculations and experimental data can be used in conjunction to aid structure analysis. Linear dichroism (LD) can be calculated using analogous methodology and has been used to establish the relative orientations of subunits in proteins and protein orientation in an environment such as a membrane. However, simple analysis of LD data is not possible, due to overlapping transitions. So coupling the calculations and experiment is an important strategy. In this paper, the use of LD for the determination of protein orientation and how these data can be interpreted with the aid of calculations, are discussed. We review methods for the calculation of CD spectra, focusing on semiempirical and ab initio parameter sets used in the matrix method. Lastly, a new web interface for online CD and LD calculation is presented.     

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.     

Specific isolation of N-terminal fragments from proteins and their high-fidelity de novo sequencing

A new method to determine N-terminal amino acid sequences of multiple proteins at low pmol level by a parallel processing has been developed. The method contains the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling with sulfosucccimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate(sulfo-NHS-SS-biotin) to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease; (4) specific isolation of N-terminal fragments of proteins by affinity capture using the biotin-avidin system; (5) de novo sequence analysis of peptides by MALDI-TOF-/MALDI-TOF-PSD mass spectrometry with effective utilization of the CAF (chemically assisted fragmentation) method.1 This method is also effective for N-terminal sequencing of each protein in a mixture of several proteins, and for sequencing components of a multiprotein complex. It is expected to become an essential proteomics tool for identifying proteins, especially when used in combination with a C-terminal sequencing method.