Pier Giorgio Righetti

Capillary isoelectric focusing: the problem of protein solubility

Abstract A whole family of protein solubilizers, compatible with native structure and maintenance of enzyme activity, is reported for preventing protein precipitation and aggregation at the p I value under conditions of very low ionic strength, as typical of isoelectric focusing methodologies. In addition to mild solubilizers proposed in the past, such as glycols (glycerol, ethylene and propylene glycols) non-detergent sulphobetaines, in concentrations up to 1 M , are found to be quite effective in a number of cass. Other common zwitterions, such as taurine and a few of the Goods buffers (e.g., Bicine, CAPS) are also quite useful in acidic pH gradients and up to pH 8. Addition of sugars, notably saccharose, sorbitol and, to a lesser extent, sorbose (20% in capillary IEF and in the 30 to 40% concentration range in gel-slab IPGs), greatly improved protein solubility in the proximity of the p I . The improvement was dramatic if these sugars were mixed with 0.2 M taurine. In the case of hydrophobic peptide antibiotics, mixtures of 6 M urea and 25% trifluoroethanol were found to markedly improve solubility. All these additives, unlike non-ionic or zwitterionic surfactant, have the advantage of remaining monomeric, i.e., of being unable to form micelles, even at concentrations > 1 M . Thus, their elimination from the protein zone can be easily accomplished by gel filtration or by centrifugation through dialysis membranes. Using these additives, capillary IEF of proteins should be now applicable to a number of difficult cases, such as the separation of mildly hydrophobic macroions.

Wall adsorption in capillary electrophoresis. Experimental study and computer simulation

Abstract A semi-quantitative model based on non-linear equilibrium chromatography coupled with diffusion-driven sample sorption at the wall was developed to account for and predict potential binding of an analyte to the wall in capillary electrophoresis. It was then used for computer simulation of sample concentration profiles corresponding to different experimental conditions (sorption kinetics, capillary length, wall capacity and initial sample concentration). The binding phenomena were also studied experimentally by means of analysis of the sample peak shape (including peak height and area). Contrary to expectations, it was found that the interaction of small monovalent cations with the charged capillary wall does not lead to strong adsorption, as the sample mass is not lost during experiment and the peak shape remains close to that which one could expect in the absence of interaction. For polycations (e.g., poly- l -histidine) at any pH above 3, sample adsorption is evident by a lack of return of the baseline to zero, after peak passage, with progressively highler levels at progressively increasing buffer pH values. Upon several runs with a polycation, the surface charge on the wall changes from negative to positive, as evidenced by reversal of electroosmotic flow. However, it was discovered that even under these last conditions, the sample-wall interaction was rather strong. The influence of NaOH washing and the addition of different substances (urea, Tween-20, sodium chloride) on adsorption was studied. The comparison between simulated results and experimental data is discussed.

Adsorption of proteins to fused-silica capillaries as probed by atomic force microscopy

Abstract In order to prove binding of proteins to the capillary wall, the inner surface of naked silica has been probed with the aid of atomic force microscopy. A large protein (ferritin, a particle of 12 nm diameter) has been left in contact with the capillary dissolved in buffers both below (pH 4.6) and above (pH 7.0) its pI (5.0–5.2) value. The capillary was then sliced lengthwise and its surface explored with the atomic force microscopy tip. Massive protein adsorption onto the naked fused-silica wall was observed, both below and above the protein pI, the thickness and extent of such deposition being proportional to the initial concentration of the protein bathing the wall. Such proteinaceous material could be largely desorbed by washing the capillary in 1 M NaOH, this process restoring the original topography of naked fused-silica. Additionally, such binding was also demonstrated electrophoretically by a displacement process which consisted of desorbing the bound ferritin by driving anionic detergent micelles (sodium dodecyl sulphate) from the cathodic compartment. Atomic force microscopy could thus become a powerful tool for probing surface adsorption also to coated capillaries, thus helping in designing better, more hydrophilic coatings.

Detection of traces of a trisulphide derivative in the preparation of a recombinant truncated interleukin-6 mutein.

A new mutein of interleukin-6, called delta 22-IL-6 Cys 3,4, characterized by the deletion of the first 22 amino acids at the N-terminal end and by the substitution of the first two cysteines (Cys23 and Cys29) with serine residues, was produced in Escherichia coli and was found to maintain the structural and functional properties of the human native form. A partially purified preparation still showed in isoelectric focusing a minor acidic component (pI 6.10) and a more basic component (pI 6.70), the native form having a pI of 6.56. This preparation was further fractionated in a multi-compartment electrolyser with isoelectric membranes, which allowed the collection of the more alkaline species for characterization. Mass spectra of the pI 6.70 form gave an additional mass of 32 atomic mass units (amu), suggesting the addition of two oxygen atoms (a potential oxidation of two methionine residues to sulphoxide). However, the five methionine residues in this higher pI form were identified after enzymatic hydrolysis and peptide mapping and were found to be in a reduced state. In addition, the pI 6.70 form was quickly converted into the native form by mild reductive treatment. On digestion and fingerprinting, the peptide from residues 50 to 65 of the pI 6.70 species (containing the only two cysteine residues of the molecule) exhibited a more hydrophobic behaviour in reversed-phase high-performance liquid chromatography and retained a mass increase of 32 amu. These experimental findings more likely suggest the addition of an extra sulphur atom to the only disulphide bridge to give an unusual protein trisulphide molecule.

Method for measuring very weak, residual electroosmotic flow in coated capillaries

Abstract Many different methods exist for measuring the velocity of electroosmotic flow in capillary electrophoresis (CE). The easiest and the most common one is photometric detection of a neutral marker. This method can be used on any CE unit, while other methods may require some modifications or special equipment. However, this method is not very suitable for measuring weak electroosmosis, since very long runs are necessary for the neutral marker to arrive at the detector. For this case, an alternatice method is proposed here. It assumes that the neutral marker is injected by means of electroosmosis, then rinsed by applying a low pressure and recorded. The resulting peak is quantified and, according to the amount of sample injected, the velocity of electroosmosis can be estimated. The proposed method is much faster than the traditional one; its duration is determined mainly by the time of electroosmotic injection. It can be used for estimation of the quality of capillary coatings, for which the residual electroosmotic flow is one of the characteristics.

Performance of a series of novel N-substituted acrylamides in capillary electrophoresis of DNA fragments

DNA separations by capillary electrophoresis in viscous solutions of novel polymers, made with Ω-hydroxyl, N-substituted acrylamides (notably N-acryloyl amino propanol, AAP and N-acryloyl amino butanol, AAB) are evaluated. Whereas in standard poly(acrylamide), at 6% concentration, the theoretical plate number (N) does not exceed 500 000, in 6% poly(AAP) N reaches 922 000 and in 6% poly(AAB) N values as high as 1 200 000 are obtained. Also, copolymers of AAP and AAB give N values in excess of 1 million plates. The two novel monomers (AAP and AAB) remain extremely stable during alkaline hydrolysis and display very good hydrophilicity, while being devoid of the noxious habit of auto-polymerization and auto-reticulation exhibited by the previous monomer of this series (N-acryloyl amino ethoxy ethanol). The reasons for such a good performance of the Ω-substituted acrylamide derivatives could be that their polymers may form hydrogen bonds via their distal -OH group during DNA separation.

‘Tunable’ positive and negative surface charges on a capillary wall: exploiting the Immobiline chemistry

The Immobiline (weak acrylamido acids and bases) chemistry has been applied to the covalent attachment of a positively (or, if needed, negatively) charged layer onto the inner surface of the silica wall. In particular, the following basic Immobilines have been used: pK 6.2, pK 7.0, pK 8.5 and pK 9.3. In order to avoid pK changes, the charged Immobilines are mixed with neutral acrylamido derivatives (in particular the highly resistant and hydrophilic N-acryloyl aminoethoxyethanol) so as to form a co-polymer having a 1:5 molar ratio (charged to neutral). The mu(eo) vs. pH curves have a slope opposite to that of a naked capillary and fan out on the pH scale following the titration curves of the different weak bases. Such chemistry allows the covalent attachment of charged species having known pK values and offering controlled charged densities on the wall. However, with the atomic force microscope, it is found that such soft coatings (whether charged or neutral) do not seem to provide complete coverage of the surface: naked patches of fused silica are found interdispersed among the polymer-coated ones. A good solution is a hybrid bonded and dynamic coating, obtained by adding short chain linear polyacrylamides to the background electrolyte. Good separations of polycations [poly(L-histidine)] and of histones are reported up to pH 5.7.

Current Gel Electrophoresis Approaches to Low-Abundance Protein Marker Discovery

Abstract Two-dimensional polyacrylamide gel electrophoresis (2-DE) is one of the oldest separation/analytical methods in biochemistry that became progressively the leading technology in proteomics. Over time it has steadily improved in sensitivity, reproducibility, and ease of manipulation. Today it remains irreplaceable in protein investigations. Although it is not the solution to all issues, it is of more interest when associated with complementary technologies capable of enhancing the detection of low-abundance species or expression modifications that intervene at the early stage of a given disease. This chapter describes 2-DE in association with a combinatorial peptide ligand library as an integrated tool to detect species that otherwise would remain undetectable.

Mutational Analysis with Capillary Electrophoresis

Analysis of DNA by capillary electrophoresis (CZE) has witnessed a remarkable growth, perhaps due to the interest spurred in the scientific community by projects like the Human Genome Sequencing and the screening for genetic defects in the human population at large. We will review here some of the main developments, with particular emphasis on the second topic, i.e. mutational analysis. Before entering this main subject, we feel an excursus in the field of separation matrices is due, since this aspect is of fundamental importance in DNA analysis, due to the fact that DNA separations are mostly driven by sieving effects in gel media (for a more detailed review about this subject, the reader is referred to Chapter 1). The unique development pertaining to CZE is the concept of sieving liquid polymers, i.e. size separations obtained not in solidified matrices (typically cross-linked Polyacrylamides and agaroses) but in liquid media containing linear or branched polymers exerting size separations with remarkable efficiencies. Such polymer solutions have unique advantages in CZE, in that they allow filling and emptying of a capillary lumen, in solutions of proper viscosity, at the end of each run, thus restoring the right experimental conditions at each subsequent run and avoiding noxius effects of repeated uses of the same matrix (e.g., carryover, buffer-ion depletion, polymer degradation). After this excursus we will give examples of the unique separation capabilities afforded by liquid Polyacrylamides (especially when adopting novel N-substituted monomers) in the 10 to 1000 bp range, i.e. an interval of great interest for DNA sequencing, oligonucleotide separations, mutational analysis.