2,318 research outputs found
Separation of peptides in isoelectric cysteic acid buffer and hydro-organic solvents (hexafluoro-2-propanol-urea).
No full textA novel amphoteric, isoelectric, acidic buffer is here reported for separation of oligo- and polypeptides by capillary zone electrophoresis: cysteic acid (Cys-A). Cys-A, at 200 mM concentration, exhibited an isoelectric point (pI) of 1.80; given a Delta pK=0.6, the pK of the carboxyl was assessed as 2.1 and the pK of the sulphate group as 1.50. At 100 mM concentration, this buffer provided an extraordinary buffering power: 140.10(-3) equiv./l per pH unit. In presence of 30% (v/v) hexafluoro-2-propanol (HFP), this buffer did not change its apparent pi value, but drastically reduced its conductivity. In Cys-A-HFP buffer, small peptides exhibited a mobility closely following the Offord equation, i.e., proportional to the ratio M-r(2/3)/Z). With addition of 4-5 M urea, there was an inversion in the mobility of some peptides, suggesting strong pK changes as an effect of urea addition. It was found that the minimum mass increment, for proper peptide separation, was Delta M-r=ca. 1%. In case of simultaneous M-r and pK changes, the minimum Delta M-r is reduced to only 0.6%, provided that a concomitant minimum Delta pK=0.08 took place. When separating large peptides (human globin chains) in 100 mM Cys-A, 30% HFP and 7 M urea, the beta-chain was found to co-elute with the alpha-chain, suggesting a subtle interplay between the helix forming (HFP) and helix breaking (urea) agents. When HFP was omitted, the original globin separation could be restored
The Search for Peptide Epitopes for Molecular Imprinting Through Bioinformatics
No full textEpitope imprinting is an effective strategy to prepare molecularly imprinted polymers (MIPs) for protein recognition. Indeed, the idea to use as a template just a fragment of the protein of interest, called the epitope, instead of the whole protein, presents some key advantages for the imprinting process, in particular: cutting the costs for MIP production and avoiding protein unfolding during the imprinting process, so to ultimately improve the quality of the stamped binding sites. How to select an epitope for the imprinting is the strategic question. Here, the bioinformatics tools to search for suitable epitopes for the imprinting process and rational tools to select the most suitable epitope are briefly introduced along with protocols for their practical use
Isoelectric focusing of protein and peptides in gel slab and in capillaries.
No full textThe present review offers a survey of modern isoelectric focusing (IEF) techniques, including the conventional one in soluble amphoteric buffers (CA-IEF) and immobilized pH gradients (IPG). In the case of CA-IEF, recent advances on its use as a first dimension for two-dimensional electrophoresis are covered. The properties of isoelectric buffers, in terms of buffering power and conductivity, are reviewed in view of their importance as background electrolytes in capillary zone electrophoresis (CZE). In the case of IPGs, examples are given of the high correlation between theoretically predicted and experimentally measured pi values. New aspects of IPGs, such as focusing in extremely alkaline pH intervals, and its interfacing with mass spectrometry, are described. In the case of capillary IEF, new aspects of the technique are described, in particular: (a) how to modulate the slope of the pH gradient for increasing resolution; and (b) how to keep proteins in solution at, and in the proximity of, the pi value. The review ends with an excursus on the use of isoelectric buffers in capillary zone electrophoretic separations. Such buffers offer unique advantages: they permit very high voltage gradients (up to 1000 V/cm) and, thus, minimize analysis times (down to a few minutes in 30-35 cm long capillaries). This results in a marked increment in resolution due to minimal diffusion-driven peak spreading. Such buffers are finding unique applications for generating peptide maps of tryptic digests of proteins and also in the analysis of intact proteins
Surface imprinted beads for the recognition of human serum albumin
No full textThe synthesis of poly-aminophenylboronic acid (ABPA) imprinted beads for the
recognition of the protein human serum albumin (HSA) is reported. In order to
create homogeneous recognition sites, covalent immobilisation of the template
HSA was exploited. The resulting imprinted beads were selective for HSA. The
indirect imprinting factor (IF) calculated from supernatant was 1.6 and the
direct IF, evaluated from the protein recovered from the beads, was 1.9. The
binding capacity was 1.4 mg/g, which is comparable to commercially available
affinity materials. The specificity of the HSA recognition was evaluated with
competitive experiments, indicating a molar ratio 4.5/1 of competitor was
necessary to displace half of the bound HSA. The recognition and binding of the
imprinted beads was also tested with a complex sample, human serum and targeted
removal of HSA without a loss of the other protein components was demonstrated.
The easy preparation protocol of derivatised beads and a good protein
recognition properties make the approach an attractive solution to analytical
and bio-analytical problems in the field of biotechnology
Isoelectric focusing in immobilized pH gradients: recent analytical and preparative developments
No full textIsoelectric focusing in immobilized pH gradients (IPG), covering both analytical and preparative aspects, is here reviewed. An extensive introduction covers the development of the technique from its inception in 1982 to present day methodology, with particular emphasis on the development of computer programs able to calculate and optimize linear and nonlinear pH gradients, spanning as much as 9 pH units, from a mixture of as many as 10 different buffering ions and titrants. The unique resolving power of IPGs is illustrated with the resolution of fetal globin chains differing by an Ala/Gly substitution in residue 75, this bringing about a minute difference in pI value of only 0.001 pH units. IPG runs, performed under denaturing conditions, allow an excellent correlation between experimental and theoretical protein pIs, to the extent that outliers were found to be polypeptide chains which had undergone post-synthetic modifications. The IPG methodology allows easy interfacing with mass spectrometry, due to the fact that proteins eluted from an IPG gel are isoionic as well as isoelectric, and thus are not contaminated by any buffer ion. The review ends with an excursus on preparative aspects of IPGs: a novel apparatus, based on the principle of isoelectric, buffering membranes, allows pilot-scale purification of r-DNA proteins to extreme purity, with recovery in a liquid vein. Isoelectric membranes have a selectivity based on a continuous titration process, and thus act as isoelectric traps for individual protein species. This same preparative apparatus can be used as a novel immobilized enzyme reactor, with superior performance compared to conventional types of reactors
An isoelectrically trapped enzyme reactor operating under an electric field
No full textMembrane enzyme reactors constitute an attempt at integrating catalytic conversion, product separation and/or concentration and catalyst recovery into a single operation. Whereas conventional membrane reactors confine an enzyme, in a free form, to one side of a membrane by size exclusion, electrostatic repulsion, or physical or chemical immobilization onto an intermediate support (gel, liposome), the membrane reactor here described is shown to operate under an entirely new principle: enzyme confinement into an isoelectric trap located in a multicompartment electrolyzer operating in an electric field. Two isoelectric membranes, having pI values encompassing both the enzyme pI and the pH of its optimum of activity, act by continuously titrating the enzyme trapped inside, thus preventing it from escaping the reaction chamber. Charged products generated by the enzyme catalysis are continuously electrophoretically transported away from the reaction chamber and collected into other chambers stacked either towards the cathodic or anodic sides. In a urease reactor, ammonia is continuously harvested towards the cathode, thus allowing >95% substrate consumption with maintenance of enzyme integrity over much longer time periods than in a batch reactor. In a trypsin reactor, casein is digested and biologically active peptides are continuously harvested in a pure form into appropriate isoelectric traps. In a third example, pure D-phenylglycine is produced from a racemate mixture, via an acylation reaction onto a cosubstrate (the ester methyl-4-hydroxyphenyl acetate), brought about by the enzyme penicillin G acylase
Rational selection of peptide epitope templates for proteins imprinting
No full textBackground
The main disadvantage in the use of proteins as templates in protein imprinting is their 3D-structure, complex and flexible, that unfolds easily. Decreasing the complexity of the protein template seems the best strategy, as demostrated by “epitope imprinting”. Yet, in all current protein MIP approaches, internal epitopes were not accessible and thus, have never been considered as templates, bringing to the loss of a huge number of potential epitope-candidates. Furthermore, in many cases conserved epitopes of diagnostic interest are located at the core of the protein, where these are not exposed to the pressure of the immune system.
Objective
Here we propose a rational method for the selection of epitopes for protein imprinting that enables targeting all epitopes, either exposed or internal. Named fingerprint imprinted polymers (FIP), in analogy with the fingerprinting analysis (the use of the constituent peptides to identify a protein).
Methods
The FIP method is based on the following steps for identifying a peptide candidate for imprinting: (I) in silico cleavage of the protein sequence by various cleaving agents; (II) selection of the resulting peptides based on length and hydrophylicity ; (III) screening of each peptide candidate against the entire protein sequences databank (e.g. UniProtKB) to eliminate candidate sequences that are not unique enough. A probability score of uniqueness is associated to the each peptide candidate, allowing the selection of unique sequences as templates.
Results
To proof the principle, NT-proBNP maker of cardiovascular risk, was chosen. The in silico analysis of NT-proBNP sequence allowed to individuate two peptide candidates, next used as templates for the preparation of NT-pro-BNP specific FIPs and tested for their ability to bind the NT-proBNP peptides in complex samples. Results indicated remarkable imprinting factor (IF 10), binding capacity of 0.5-2 mg/g, in line with many affinity materials, ability to rebind the 40% of template in a complex sample, composed of the whole digests of NT-proBNP.
Conclusions
Results supported the validity of the method here proposed. Key point is the fragmentation of the protein into peptides. The advantage is the ability to circumvent the issue of the folding of the protein
Isoelectric focusing in immobilized pH gradients: an update.
No full textThe latest trends on isoelectric focusing (IEF) in immobilized pH gradients (IPG) are here reviewed. The major advances on IPG technologies have been made when interfacing this technique with sodium dodecyl sulfate-polyacrylamide gel electrophoresis to produce two-dimensional (2-D) maps. Previous 2-D maps were routinely performed using conventional IEF as a first dimension, which typically resulted in poor reproducibility of spot position. With IPGs, correlation between experimental and calculated protein pI values is as good as +0.01 to 0.02 pH units. A new software has also been released, permitting easy calculation and optimization of linear, concave and convex exponential gradients, even in very complex recipes utilizing all ten Immobiline chemicals. It has also been proven that IPGs can be interfaced with mass spectrometry, thus obtaining a novel 2-D map with the best of pI measurements in the first dimension coupled with the best of mass determination in the second dimension. Recently, it has been shown that IPGs can be exploited to charter forbidden grounds, with the creation of non-linear pH gradients covering the extreme alkaline pH 10-12 gradient. In such basic regions, excellent steady-state patterns of histones and subtilisin mutants have been reported. Different families of histones could be mapped not only in this pH 10-12 interval, but also in 2-D maps exploiting this very alkaline gradient in the first dimension. Although the IPG technique is now a trouble-free, user-friendly technique, some annoying artefacts, producing severe protein smears and precipitation, were very recently reported, but found to be linked to some commercial Immobiline preparations containing up to 5% oligomers. Better quality control on the part of the company producing such chemicals should eliminate even this last source of troubles
RULES TO PREPARE PEPTIDE-IMPRINTED NANOGELS WITH HIGH-AFFINITY BINDING SUITABLE FOR SENSING AND ASSAYS BY PRECIPITATION POLYMERIZATION
No full textMolecular imprinting is a technique for preparing polymeric scaffolds (Molecularly Imprinted Polymers, MIPs) that function as synthetic receptors and show affinity and selectivity towards a target analyte. The most attractive characteristic of MIPs is the possibility to tailor the binding selectivity so to gain recognition levels on the par of biological receptors. When MIPs are downsized to the nanoscale (nanoMIPs), they show an increase in the number of accessible imprinted binding cavities per material weight and an enhanced molecular recognition ability, leading to faster binding kinetics, higher affinity and selectivity, thus strengthening the resemblance to antibodies and natural receptors. Being the recognition properties of the nanoMIPs strictly correlated to the effective formation of the imprints in the chosen synthetic conditions, a deeper comprehension of the polymerization at the nanoscale is required. In order to fill this lack, we studied the best conditions to form imprints at the nanoscale when the synthesis occurs by a precipitation polymerization protocol by means of an one-pot synthesis via free radical initiation in aqueous solution, using as target analyte the peptide of Troponin I, clinical marker of cardiac failure. By exploring a range of monomers combinations, polyacrylamide-based MIP nanogels having homogeneous nano-dimensions and a low number of binding sites per nanoparticle were synthesized. To this purpose, we evaluated the influence of the monomer composition and the total monomers to template molar ratio on the hydrodynamic sizes and on the recognition properties, respectively, defining the conditions to tune the nanoMIP dimensions (from 60 to >600 nm) and to improve the efficacy of the imprinting process. In the light of the achieved results, the present work contributes to define the best conditions to obtain imprinted peptides at the nanoscale and impact on the production of synthetic recognition materials suitable for sensing and assays
Molecularly imprinted polymers by epitope imprinting: a journey from molecular interactions to the available bioinformatics resources to scout for epitope templates
No full textThe molecular imprinting of proteins is the process of forming biomimetics with entailed protein-recognition by means of a template-assisted synthesis. Protein-imprinted polymers (pMIPs) have been successfully employed in separations, assays, sensors, and imaging. From a technical point of view, imprinting a protein is both costly, for protein expression and purification, and challenging, for the preservation of the protein's structural properties. In fact, the imprinting process needs to guarantee the preservation of the same protein three-dimensional conformation that later would be recognized. So far, the captivating idea to imprint just a portion of the protein, i.e., an epitope, instead of the whole, proved successful, offering reduced costs, compatibility with many synthetic conditions (solvents, pH, temperatures), and fine-tuning of the peptide sequence so to target specific physiological and functional conditions of the protein, such as post-translational modifications. Here, protein-protein interactions and the biochemical features of the epitopes are inspected, deriving lessons to prepare more effective pMIPs. Epitopes are categorized in linear or structured, immunogenic or not, located at the protein's surface or buried in its core and the imprinting strategies are discussed. Moreover, attention is given to freely available online bioinformatics resources that might offer key tools to gain further rationale amid the selection process of suitable epitopes templates
- …
