Alessandra Bossi

Substitution of Antibodies and Receptors with Molecularly Imprinted Polymers in Enzyme-Linked and Fluorescent Assays

A new technique for coating microtitre plates with molecularly imprinted polymers (MIP), specific for low-molecular weight analytes (epinephrine, atrazine) and proteins is presented. Oxidative polymerization was performed in the presence of template; monomers: 3-aminophenylboronic acid (APBA), 3-thiopheneboronic acid (TBA) and aniline were polymerized in water and the polymers were grafted onto the polystyrene surface of the microplates. It was found that this process results in the creation of synthetic materials with antibody-like binding properties. It was shown that the MIP-coated microplates are particularly useful for assay development. The high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive alternative to conventional antibodies or receptors used in enzyme linked immunosorbent assay (ELISA).

Fingerprint-imprinted polymer: rational selection of peptide epitope templates for the determination of proteins by molecularly imprinted polymers.

The pool of peptides composing a protein allows for its distinctive identification in a process named fingerprint (FP) analysis. Here, the FP concept is used to develop a method for the rational preparation of molecularly imprinted polymers (MIPs) for protein recognition. The fingerprint imprinting (FIP) is based on the following: (1) the in silico cleavage of the protein sequence of interest with specific agents; (2) the screening of all the peptide sequences generated against the UniProtKB database in order to allow for the rational selection of distinctive and unique peptides (named as epitopes) of the target protein; (3) the selected epitopes are synthesized and used as templates for the molecular imprinting process. To prove the principle, NT-proBNP, a marker of the risk of cardiovascular events, was chosen as an example. The in silico analysis of the NT-proBNP sequence allowed us to individuate the peptide candidates, which were 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 an imprinting factor, IF, of ~10, a binding capacity of 0.5-2 mg/g, and the ability to rebind 40% of the template in a complex sample, composed of the whole digests of NT-proBNP.

Capillary electrophoresis of peptides and proteins in isoelectric buffers: an update.

Capillary electrophoresis in acidic, isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and little interaction of macromolecules with the untreated wall occurs. In addition, due to the low conductivity of quasi‐stationary, isoelectric buffers, high‐voltage gradients can be applied (up to 800 V/cm) permitting fast peptide analysis with a high resolving power due to minimal diffusional peak spreading. Four such buffers are here described: cysteic acid (Cys‐A, pI 1.85), iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). A number of applications are reported, ranging from food analysis to the study of folding/unfolding transitions of proteins.

Isoelectric focusing in immobilized pH gradients: an update

The 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.

Production of D‐phenylglycine from racemic (D,L)‐phenylglycine via isoelectrically‐trapped penicillin G acylase

Penicillin G acylase (PGA) is exploited for producing pure D-phenylglycine from a racemate mixture, via an acylation reaction onto a cosubstrate, the ester methyl-4-hydroxyphenyl acetate. The reaction, when carried in a batch, is severely hampered by the reverse process, by which the product, 4-hydroxyphenylacetyl-(L)phenyl glycine, upon consumption of L-phenylglycine, is converted by the enzyme back into free substrate and 4-hydroxyphenyl acetic acid via lysis of the amido bond. To prevent this noxious reaction, a multicompartment electrolyzer with isoelectric membranes (MIER) is used as enzyme reactor, operating in an electric field. PGA is trapped between pI 5.5 and pI 10.5 membranes, together with an amphoteric, isoelectric buffer (lysine). As the 4-hydroxyphenylacetyl-(L)phenyl glycine product is formed, it vacates the reaction chamber by electrophoretic transport and is collected close to the anode, in a chamber delimited by pI 2.5 and 4.0 membranes. The same fate occurs to the free acid 4-hydroxyphenyl acetic acid, formed upon spontaneous (and enzyme-driven) hydrolysis of the methyl ester in the reaction chamber. These combined processes leave behind, in the enzyme reaction chamber, the desired product, pure D-phenylglycine. The advantages of the MIER reactor over batch operations: the consumption of the L-form in the racemate is driven to completion and the enzyme is kept in a highly stable form, maintaining 100% activity after one day of operation, during which time the PGA enzyme, in the batch reactor, has already lost >75% catalytic activity.

Capillary electrophoresis coupled to biosensor detection

The present review highlights some modern aspects of biosensor revelation, a detection method which has already found a large number of applications in healthcare, food industry and environmental analysis. First, the concept of bio-recognition, which is at the heart of biosensor technology, is discussed, with emphasis on host-guest-like recognition mechanisms. This detection device has been successfully coupled, in its first applications, to chromatographic columns, which allow a high resolution of complex mixtures of analytes prior to interaction with the biosensing unit. The properties of the transducing elements, which should generate a signal (e.g., electrochemical, thermal, acoustic, optical) of proper intensity and of relative fast rise, are additionally evaluated and discussed. The review then focuses on potential applications of biosensing units in capillary electrophoresis (CE) devices. CE appears to be an excellent separation methodology to be coupled to biosensor detection, since it is based on miniaturized electrophoretic chambers, fast analysis times, complete automation in sample handling and data treatment and requires extremely small sample volumes. Although only a few applications of CE-based biosensors have been described up to the present, it is anticipated that this hyphenated technique could have a considerable expansion in the coming years.

Proteomic analysis of dopamine and α-synuclein interplay in a cellular model of Parkinson’s disease pathogenesis

Altered dopamine homeostasis is an accepted mechanism in the pathogenesis of Parkinson’s disease. α‐Synuclein overexpression and impaired disposal contribute to this mechanism. However, biochemical alterations associated with the interplay of cytosolic dopamine and increased α‐synuclein are still unclear. Catecholaminergic SH‐SY5Y human neuroblastoma cells are a suitable model for investigating dopamine toxicity. In the present study, we report the proteomic pattern of SH‐SY5Y cells overexpressing α‐synuclein (1.6‐fold induction) after dopamine exposure. Dopamine itself is able to upregulate α‐synuclein expression. However, the effect is not observed in cells that already overexpress α‐synuclein as a consequence of transfection. The proteomic analysis highlights significant changes in 23 proteins linked to specific cellular processes, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis, and neuronal plasticity. A bioinformatic network enrichment procedure generates a significant model encompassing all proteins and allows us to enrich functional categories associated with the combination of factors analyzed in the present study (i.e. dopamine together with α‐synuclein). In particular, the model suggests a potential involvement of the nuclear factor kappa B pathway that is experimentally confirmed. Indeed, α‐synuclein significantly reduces nuclear factor kappa B activation, which is completely quenched by dopamine treatment.

Protein purification in multicompartment electrolyzers with isoelectric membranes

Preparative purification of proteins under isoelectric conditions is reviewed, with particular regard to novel equipment, a multicompartment electrolyzer with isoelectric membranes, which can capture any desired protein into an isoelectric trap as the sole, ultra-pure component. This novel machine is based on the Immobiline chemistry, i.e. the novel generation of non-amphoteric buffers, based on the chemistry of acrylamides, which can be insolubilized onto polyacrylamide supports. After a description of the instrument and of its performance, a number of protein purification protocols are described, leading to truly homogeneous (by the most stringent criterion of surface charge) protein fractions. Such a high charge purity has been found to be often a fundamental prerequisite for the growth of protein crystals. Interfacing the electrolyzer with mass spectrometry has permitted the decoding of the structure of minor components generated from a parental molecule, especially ones having a higher pI. It was found that these species were often generated either by proteolytic cleavage or by the formation of a trisulphide bridge between two Cys residues. A unique application of the electrolyzer is finally described: its use as an immobilized enzyme reactor under an electric field. The performance of this reaction is outstanding, in that the kinetic parameters of the immobilized enzyme are identical to those of a free enzyme form.

Continuous Enzymatic Hydrolysis of β‐Casein and Isoelectric Collection of Some of the Biologically Active Peptides in an Electric Field

Among the milk proteins, bovine β‐casein has the peculiarity of containing in its sequence some peptides liable to interfere in mineral nutrition and some peptides with opioid (casomorphines), antihypertensive, and immunomodulatory activities. In this work we propose a novel type of multicompartment enzyme reactor, operating under an electric field, for the continuous hydrolysis of milk proteins such as β‐casein. The enzyme trypsin is trapped, with zwitterionic buffering ions and its substrate β‐casein, in solution between two isoelectric membranes having pI values encompassing the isoelectric point of the enzyme. Additionally, β‐casein is captured inside the same reaction chamber with the aid of sieving membranes, since its pI is too far away from the pI of trypsin. This setup permits the continuous operation at the pH of optimum of activity. The peptides, arising from tryptic hydrolysis of β‐casein, are removed under the influence of the electric field and collected in different chambers in which they are isoelectric and isoionic as well. The purity of the peptides collected is ascertained by capillary zone electrophoresis and their identity confirmed by N‐terminal sequencing and MALDI‐TOF mass spectrometry. This setup allows continuous harvesting of some biologically‐active peptides in a pure form. The major advantages of such a reactor system over conventional batch reactors are the great increase in enzyme utilization efficiency and the overall reactor productivity.

Separation of peptides in isoelectric cysteic acid buffer and hydro-organic solvents (hexafluoro-2-propanol-urea)

Abstract A 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 m M concentration, exhibited an isoelectric point (p I ) of 1.80; given a Δp K =0.6, the p K of the carboxyl was assessed as 2.1 and the p K of the sulphate group as 1.50. At 100 m M 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 p I 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 p K changes as an effect of urea addition. It was found that the minimum mass increment, for proper peptide separation, was Δ M r =ca. 1%. In case of simultaneous M r and p K changes, the minimum Δ M r is reduced to only 0.6%, provided that a concomitant minimum Δp K =0.08 took place. When separating large peptides (human globin chains) in 100 m M Cys-A, 30% HFP and 7 M urea, the β-chain was found to co-elute with the α-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.