Hanno Stutz

Protein attachment onto silica surfaces--a survey of molecular fundamentals, resulting effects and novel preventive strategies in CE.

This review addresses the fundamentals governing the adsorption of individual protein molecules onto the surface of fused‐silica capillaries, the protein aggregation to adsorbate clusters and their final accretion to monolayers with subsequent stratification to protein multilayers. The attention in CE protein separation has primarily been focused on (i) tuning the BGE including the buffer type, ionic strength, pH and additives, (ii) tailored post‐rinse procedures to detach adhered protein residues and (iii) the optimization of capillary wall shielding in order to reduce protein attachment. Improvements in protein separation as well as related adverse effects are mainly discussed on the basis of parameters known to become deteriorated in case of protein adhesion, e.g. repeatability of the EOF and of migration times, peak width, theoretical plate numbers, resolution and asymmetry factor. However, knowledge of the molecular principles controlling protein adsorption onto silica surfaces is indispensable for separation optimization. Furthermore, it facilitates troubleshooting and the interpretation of undesired concomitant phenomena. This review comprehensively discusses protein adsorption models derived from surface chemistry primarily in terms of their relevance for CE, clearly showing that the adsorption process in its complexity is only partially revealed by models, which address single or binary protein solutions. In a further section theoretical concepts and surface models are related to surface phenomena encountered in CE. The final part of the review surveys recent concepts for prevention of protein adhesion, thereby addressing capillary treatment, favorable buffer types, dynamic and adhesive semi‐permanent coating strategies covering the literature from 2000–2008.

Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening.

BACKGROUND The incidence of allergic diseases in developed countries has been increasing constantly in the last 2 decades. The only curative treatment available thus far is specific immunotherapy (SIT). The problematic side effects that can occur during SIT with allergen extracts led to the search for safer alternatives, such as recombinant hypoallergenic proteins with reduced allergenic potential and preserved T-cell immunogenicity. OBJECTIVE We created hypoallergenic variants of the allergens Bet v 1a and Phl p 5b by using in silico mutation and screening and characterized the biochemical and immunologic properties of selected mutant proteins. METHODS Knowledge-based potentials were used to estimate structural changes of the protein structures under sequence variation. IgE antibodies and their cross-linking capacity were determined by using a basophil release assay, binding of human birch pollen-specific IgE was investigated by means of ELISA, and ELISPOT assays were performed to examine the T-cell immunogenicity. RESULTS Selected mutated proteins showed significantly reduced IgE-binding and cross-linking ability but retained their T cell-stimulating properties. Immunization with the hypoallergenic mutants induced blocking antibodies against murine and human IgE epitopes. CONCLUSION In silico calculation and selection of mutations that render a protein hypoallergenic represents a novel and rapid tool to create candidate molecules for SIT with recombinant allergens.

Successive multiple ionic polymer layer coated capillaries in the separation of proteins - recombinant allergen variants as a case study.

A successive multiple ionic polymer layer (SMIL) coating consisting of two pairs of poly(diallyldimethylammonium chloride) and dextran sulfate (DS) layers was applied for the separation of recombinant products of the major birch pollen allergen Betula verrucosa (Bet v 1a). The combination with volatile ammonium bicarbonate buffer at pH 6.70 offers the possibility for future MS hyphenation. The negative net charge of allergens required DS as terminal SMIL layer. The EOF was accelerated from 3.17×10−8 m2 V−1 s−1 in uncoated to 4.52×10−8 m2 V−1 s−1 in SMIL capillaries. Fresh prepared SMIL capillaries showed slight EOF acceleration due to gradual re‐organization of SMIL structure until stabilization was achieved. Dry storage of SMIL capillaries prevented fluctuations in EOF and migration times and improved coating durability. However, the gradual reconstitution of entangled SMIL layers affected efficiency, but was cured by a 10 mmol/L NaOH rinsing step. Durability of SMIL capillaries in MS‐applicable dimension was confirmed for > 70 runs and in total 42 h of voltage application with average intra‐day precision of 0.22 and 0.79% and inter‐day‐precision of 0.91 and 1.17% for migration times of EOF and Bet v 1a, respectively. Final SMIL coating allowed for the separation of Bet v 1a, a hypoallergenic isoform and carbamylated variants with 150 000–685 000 plates.

Reversible biofunctionalization of surfaces with a switchable mutant of avidin.

Label-free biosensors detect binding of prey molecules (″analytes″) to immobile bait molecules on the sensing surface. Numerous methods are available for immobilization of bait molecules. A convenient option is binding of biotinylated bait molecules to streptavidin-functionalized surfaces, or to biotinylated surfaces via biotin-avidin-biotin bridges. The goal of this study was to find a rapid method for reversible immobilization of biotinylated bait molecules on biotinylated sensor chips. The task was to establish a biotin-avidin-biotin bridge which was easily cleaved when desired, yet perfectly stable under a wide range of measurement conditions. The problem was solved with the avidin mutant M96H which contains extra histidine residues at the subunit-subunit interfaces. This mutant was bound to a mixed self-assembled monolayer (SAM) containing biotin residues on 20% of the oligo(ethylene glycol)-terminated SAM components. Various biotinylated bait molecules were bound on top of the immobilized avidin mutant. The biotin-avidin-biotin bridge was stable at pH ≥3, and it was insensitive to sodium dodecyl sulfate (SDS) at neutral pH. Only the combination of citric acid (2.5%, pH 2) and SDS (0.25%) caused instantaneous cleavage of the biotin-avidin-biotin bridge. As a consequence, the biotinylated bait molecules could be immobilized and removed as often as desired, the only limit being the time span for reproducible chip function when kept in buffer (2-3 weeks at 25 °C). As expected, the high isolectric pH (pI) of the avidin mutant caused nonspecific adsorption of proteins. This problem was solved by acetylation of avidin (to pI < 5), or by optimization of SAM formation and passivation with biotin-BSA and BSA.

Confirmation of immuno-reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity-CIEF.

Affinity‐CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno‐chemically. For this purpose mAbs of the IgG‐type have been produced in‐lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS‐PAGE, MALDI‐TOF‐MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98–7.09 and 6.78–7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 μmol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno‐chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag–antibody complexes and Ag variants coexisting in one sample.

Capillary zone electrophoretic determination of hydroxymetabolites of atrazine in potable water using solid-phase extraction with Amberchrom resins

A CZE-separation system for hydroxyatrazine, desisopropylhydroxyatrazine, desethylhydroxyatrazine and ammeline is presented that allows their determination in environmental waters in the subμgL−1 domain after solid-phase extraction/enrichment with styrene-divinylbenzene and methacrylate macroporous (Amberchrom) resins. The CZE separation and determination system uses the stacking effect and an increased light path for UV-detection (bubble cell). The etching of the bubble cell was done in our laboratory. A ruggedness check of the CZE-system revealed the resolution of theN-dealkylated metabolites to be sensitive to pH, temperature and current variations as well as to changes of capillary dimensions. Fine-tuning of the separation is performed by variation of the current intensity in the constant-current mode. Recoveries from fortified tap-water samples were 95% for hydroxyatrazine, 70% for desethylhydroxyatrazine and approximately 20% for desisopropylhydroxyatrazine at the 0.2 μg L−1 concentration level. Due to its high polarity, ammeline is only slightly adsorbed on Amberchrom resins and cannot be enriched using these materials.

Separation and characterization of nitrated variants of the major birch pollen allergen by CZE-ESI-μTOF MS

A CZE‐ESI‐TOF MS method has been optimized for the separation and identification of nitrated variants of the major birch pollen allergen from Betula verrucosa, isoform 1a (Bet v 1a). In‐house nitration of recombinant Bet v 1a was done by peroxynitrite. As a BGE, 10 mmol/L ammonium bicarbonate with pH 7.50 provided best resolution. Nebulizer gas pressure and sheath liquid flow rate of 0.4 bar and 6 μL/min, respectively, maintained CZE selectivity and constituted stable electrospray conditions. A sheath liquid composition of 75% v/v methanol with 0.1% v/v formic acid in ultrapure water resulted in highest signal intensities. Alternatively, methanol could be replaced by 50% v/v isopropanol. Two modified allergen products derived from reaction mixtures that contained different amounts of the nitration reagent were compared by the elaborated CZE‐ESI‐TOF MS method. Up to twelve different Bet v 1a variants with one‐ to sixfold nitration could be distinguished. Several allergen fractions of equivalent nitration grade were resolved. Their different migration times indicate site‐specific nitration with concomitant differences in pI and maybe also in hydrodynamic radius. The method allows for a characterization of in‐house nitrated allergen samples that are intended for testing the postulated enhanced allergenicity of nitrated Bet v 1a variants.

Nitration of β-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice.

Background We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins. Objective The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy. Design BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen β-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood. Results A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation. Conclusion Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.

Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants

One pressure and three chemical mobilization strategies have been optimized and tested for two-step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1-10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3-10 with Pharmalyte 5-6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5-6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. l-Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. l-Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed.

Separation of 18 modern plant protectants using cyclodextrin modified micellar electrokinetic chromatography including an ion-pairing reagent

A cyclodextrin-modified micellar electrokinetic chromatography separation for 18 different pesticides (metsulfuron-methyl, rimsulfuron, thifen-sulfuron-methyl, desethylatrazine, desisopropylatrazine, atrazine, simazine, terbuthylazine, 2,4-D, MCPP, MCPB, dicamba, linuron, alachlor, metolachlor, orbencarb, propiconazole, prochloraz) from eight different substance classes with very varying chemical and physical properties is presented. In particular acid-base characteristics and water-octanol distribution coefficients diverge in wide ranges. β-cyclodextrin was successfully employed as a modifier in separating the hydrophobic analytes. Peak distortion of some neutral analytes, which is devoted to the methanol content of the sample zone, was reduced by increasing the SDS concentration. Methanol in the sample is necessary for a better solubility of the hydrophobic pesticides. Few optima of SDS concentration exist allowing the separation of sulfonylureas as well as phenoxy acids and hydrophobic pesticides. An improved resolution of the sulfonylureas was achieved with tetramethylammonium chloride, which was used as an ion-pairing reagent.Final operating conditions for the separation of all these plant protectant compounds by MEKC in just one single run are a 27 mmolL−1 phosphate buffer, pH 8.03, with 95 mmolL−1SDS, 5 mmolL−1 β-cyclodextrin and 10 mmol L−1 tetramethylammonium-chloride.