The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent-tryptophan interaction.

BACKGROUND: The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding. METHODS: Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy. RESULTS: Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers [19]. This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations <10nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations. GENERAL SIGNIFICANCE: Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1-10nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.

Seroreactivity to E. coli outer membrane protein C antibodies in active inflammatory bowel disease; diagnostic value and correlation with phylogroup B2 E. coli infection.

BACKGROUND: Several serologic tests, including anti-outer membrane porin C antibody (Omp C), are used for screening and as marker of disease course in inflammatory bowel diseases (IBD). Our aim was to investigate possible differences in Omp C level in patients with active and inactive IBD compared to controls. METHODS: All blood samples were tested for Omp C. Disease activity was evaluated by Harvey Bradshaw Index, Simple Clinical Activity Index and Modified Pouchitis Disease Activity Index. RESULTS: Blood samples were collected from 113 patients and 60 controls. Patients with active IBD did not have a higher level of Omp C than patients in remission. Surprisingly, in patients with active Crohn's disease a significantly lower level of Omp C was found compared with patients with inactive Crohn's disease (p < 0.05). All other groups among patients with IBD did have a significantly higher level of Omp C, compared with controls, including patients with acute gastroenteritis (p < 0.05). Although IBD patients with phylogroup B2 E. coli cultured from their fecal samples, were more likely to have a positive Omp C test (p < 0.05), this could not explain the low Omp C level in the subgroup of patients with active Crohn's disease. CONCLUSIONS: Omp C titer was not raised in patients with active IBD compared with patients in remission. In addition, there was no difference in Omp C level in patients with active Crohn's disease compared with controls. These observations do not support the use of Omp C serology testing, either in disease activity assessment, or in screening for active Crohn's disease.

A learning-based approach towards the data-driven predictive control of combined wastewater networks - An experimental study.

Smart control in water systems aims to reduce the cost of infrastructure expansion by better utilizing the available capacity through real-time control. The recent availability of sensors and advanced data processing is expected to transform the view of water system operators, increasing the need for deploying a new generation of data-driven control solutions. To that end, this paper proposes a data-driven control framework for combined wastewater and stormwater networks. We propose to learn the effect of wet- and dry-weather flows through the variation of water levels by deploying a number of level sensors in the network. To tackle the challenges associated with combining hydraulic and hydrologic modelling, we adopt a Gaussian process-based predictive control tool to capture the dynamic effect of rain and wastewater inflows, while applying domain knowledge to preserve the balance of water volumes. To show the practical feasibility of the approach, we test the control performance on a laboratory setup, inspired by the topology of a real-world wastewater network. We compare our method to a rule-based controller currently used by the water utility operating the proposed network. Overall, the controller learns the wastewater load and the temporal dynamics of the network, and therefore significantly outperforms the baseline controller, especially during high-intensity rain periods. Finally, we discuss the benefits and drawbacks of the approach for practical real-time control implementations.

Optimization of antibody immobilization for on-line or off-line immunoaffinity chromatography.

The covalent immobilization of antibodies to solid supports for immunoaffinity (IA) purification is widely used in the biological sciences. However, relative immobilization yields, immobilization stability, and antigen-binding capacity vary significantly with the antibody and protocol used. A systematic study was conducted to determine the most versatile antibody immobilization method for use in on-line and off-line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We evaluated a method to survey optimal elution conditions and estimated immobilization yields, matrix stability, antigen binding capacities, and antigen recovery of different IA matrices. Some mAbs were sensitive to aminogroup-based immobilization, i.e., losing antigen binding capabilities after immobilization especially using epoxy chemistry. In general, the most optimal covalent antibody immobilization for on-line IA-LC-MS was achieved using aminogroup immobilization of intact antibodies by epoxy- or aldehyde-activated POROS R20-matrices and in some cases by chemical crosslinking to Protein G-POROS. Protein G-based matrices are very stable showing essentially no decline in performance after 50 application-wash-elution-reequilibration cycles and being easily prepared within 2-3 h of working time with a typical antibody coupling yield of above 80%. In off-line applications where constant flow conditions are not used, covalent crosslinking onto Protein G-POROS or Protein G-agarose is to be recommended.

Extraction and identification of electroimmunoprecipitated proteins from agarose gels.

A method for the identification of protein antigens captured in electroimmunoprecipitates was developed. Different antigen-antibody precipitates were generated by agarose gel immunoelectrophoresis. The immunoprecipitates were excised and various methods for extracting and dissociating the precipitates were systematically studied by analyzing for protein components of the extracts using peptide mass fingerprinting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal recovery of antigen was obtained by 24-h extraction at 37 degrees C using a minimal volume of 0.06 M Tris-HCl, 10% SDS (pH 7). This simple and robust method is useful for the characterization of antibody specificity. It can also be used to identify antigens generating unknown precipitates in crossed immunoelectrophoresis with polyspecific antisera, including human IgG-antigen complexes electroimmunoprecipitated by secondary antibodies. Thus, the method may prove useful as an additional technique in biomarker discovery.

Analysis of proteins in solution using affinity capillary electrophoresis.

Analysis of protein interactions by means of capillary electrophoresis (CE) has unique challenges and rewards. The choice of analysis conditions, especially involving electrophoresis buffers, are crucial and not universal for protein analysis. If conditions for analysis can be worked out, it is possible to utilize CE quantitatively and qualitatively to characterize protein-ligand binding involving unmodified molecules in solution and taking place under physiological conditions. This chapter deals with the most important practical considerations in capillary electrophoretic affinity approaches, affinity CE (ACE). The text emphasizes the most critical factors for successful analyses and has application examples illustrating various types of information offered by ACE-based studies. Also included are step-by-step accounts of the two main classes of experimental design: the pre-equilibration ACE (in the form of CE-frontal analysis (CE-FA)) and mobility shift ACE together with examples of their use. The ACE approaches for binding assays of proteins should be considered when the biological material is scarce, when any kind of labeling is not possible or desired, when the interacting molecules are the same size and when rapid and simple method development is a priority.

Direct analysis of 15N-label in amino and amide groups of glutamine and asparagine.

A novel method for on-line determination of the amount and position of 15N-labeling in complex mixtures of amino acids is presented. Underivatized amino acids were analyzed by ion-pair chromatography in combination with mass spectrometry. This enables the direct determination of the 15N label distribution. The fragmentation pathways of the nitrogen moieties of glutamine (Gln) and asparagine (Asn) were studied in detail using all mono 15N isotopomers, which led to a method for differentiating between 15N-amide and 15N-amino labeling. The fragmentation involving the amino and amide groups of Gln led to distinct ion structures. The equivalent fragmentation pattern was not observed for Asn. Instead, the amide group of Asn was eliminated as HNCO in a secondary process. The developed analytical method was evaluated by analysis of a range of standard mixtures taking into account different levels of 15N abundance and distribution between the amino and amide groups. The detection limit (3 SD) for the presence of a 15N label was 0.7 and 1.0% for Gln and Asn, respectively. The determination of the positional labeling follows a nonlinear function. A representative example at 30% 15N was used as a benchmark resulting in average relative standard deviations of 2.7 and 15% for Gln and Asn, respectively. The corresponding expectation windows for the positional labeling were found to be 2 and 12%, respectively.

Unravelling in vitro variables of major importance for the outcome of mass spectrometry-based serum proteomics.

The use of mass spectrometry (MS) for analysing low-molecular weight proteins and peptides from biological fluids has a great, yet not fully realized, potential for biomarker discovery. To prune MS-data as much as possible for non-relevant non-biological variation the development of standardized protocols for handling and processing the samples before MS and adjusting data after MS to compensate for method-induced variability are warranted. This calls for knowledge about how different variables contribute to MS-based proteome analyses. In addition, identification of the peptides involved in pre-analytical variation will be helpful in evaluating the clinical significance of predictive models derived from MS data. Using human sera, extraction by weak cation-exchange magnetic beads, and analysis by MALDI-TOF MS we here evaluated pre-analytical variation and identify peptides involved in this. The influences of humidity, temperature, and time for preparation of sera on spectral changes were evaluated. Also, the reproducibility of the methods and the effect of a baseline correction procedure were examined. Low temperatures, short handling times, and a baseline correction procedure minimize the contribution of artifacts to sample variability as observed by MS. The complement split product C3f and fragments thereof appear to be sensitive indicators of sample handling induced modifications. Other peptides that are indicative of such variability are fibrin and kininogen fragments. Using strict experimental guidelines as well as standardized sample collection procedures it is possible to obtain reproducible peak intensities and positions in serum mass profiling using magnetic bead-based fractionation and MALDI-TOF MS.

Proteinase 3 carries small unusual carbohydrates and associates with αlpha-defensins.

The neutrophil granulocyte is an important first line of defense against intruding pathogens and it contains a range of granules armed with antibacterial peptides and proteins. Proteinase 3 (PR3) is one among several serine proteases of the azurophilic granules in neutrophil granulocytes. Here, we characterize the glycosylation of PR3 and its association with antimicrobial human neutrophil peptides (HNPs, α-defensins) and the effect of these on the mechanism of inhibition of the major plasma inhibitor of PR3, α1-antitrypsin. The glycosylation of purified, mature PR3 showed some heterogeneity with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with α-defensins and oligomers hereof. Irreversible inhibition of PR3 by α1-antitrypsin did not affect its association with defensins. Other proteins from neutrophil granules were also found to be associated with defensins, whereas purified plasma proteins did not carry defensins. These results point to a role of defensins in controlling and targeting the activity of neutrophil granule proteins.

Development and preclinical safety evaluation of a new therapeutic HIV-1 vaccine based on 18 T-cell minimal epitope peptides applying a novel cationic adjuvant CAF01.

Therapeutic immunization of HIV-1-infected individuals with or without anti-retroviral therapy is a new promising disease prevention. To induce a new cytotoxic T(CD8) lymphocyte (CTL) immunity during chronic HIV-1 infection 15 infrequently targeted but conserved HLA-supertype binding CTL epitopes from Gag, Pol, Nef, Env, Vpu and Vif were identified. The 15 T(CD8) and three T(CD4) helper peptides were GMP synthesised and formulated with a new adjuvant CAF01 which is a synthetic two-component liposomic adjuvant comprising the quaternary ammonium dimethyl-dioctadecyl-ammonium (DDA) and the immune modulator trehalose 6,6'-dibehenate (TDB). Using IFN-γ ELISPOT assay, T-cell immune induction by the vaccine was found to both CD4 and CD8 T-cell restricted peptides in HLA-A2 transgenic mice. Comprehensive toxicity studies of the CAF01 adjuvant-alone and together with different vaccines showed that CAF01 when tested at human dose levels was safe and well tolerated with only local inflammation at the site of injection and no systemic reactions. No pharmacological safety issues were observed in Beagle dogs. The HIV-1 vaccine toxicity study in the Göttingen Minipig(®) showed no systemic toxicity from five repetitive i.m. injections, each with a 2-week interval, of either the 18 HIV-1 peptide antigen solution (AFO18) or the AFO18-CAF01, in which the 18 HIV-1 peptides were formulated with the CAF01 adjuvant. Distinct inflammatory responses were observed in the injected muscles of the AFO18-CAF01 vaccine treated animals as a result of the immune stimulating effect of the adjuvant on the vaccine. The results of the toxicity studies provide optimism for phase I clinical trials evaluating the therapeutic HIV-1 T-cell vaccination approach using multiple subdominant minimal epitope peptides applying the novel cationic adjuvant CAF01.

Recent applications of affinity interactions in capillary electrophoresis.

Systems biology depends on a comprehensive assignment and characterization of the interactions of proteins and polypeptides (functional proteomics) and of other classes of biomolecules in a given organism. High-capacity screening methods are in place for ligand capture and interaction screening, but a detailed dynamic characterization of molecular interactions under physiological conditions in efficiently separated mixtures with minimal sample consumption is presently provided only by electrophoretic interaction analysis in capillaries, affinity CE (ACE). This has been realized in different fields of biology and analytical chemistry, and the resulting advances and uses of ACE during the last 2.5 years are covered in this review. Dealing with anything from small divalent metal ions to large supramolecular assemblies, the applications of ACE span from low-affinity binding of broad specificity being exploited in optimizing selectivity, e.g., in enantiomer analysis to miniaturized affinity technologies, e.g., for fast processing immunoassay. Also, approaches that provide detailed quantitative characterization of analyte-ligand interaction for drug, immunoassay, and aptamer development are increasingly important, but various approaches to ACE are more and more generally applied in biological research. In addition, the present overview emphasizes that distinct challenges regarding sensitivity, parallel processing, information-rich detection, interfacing with MS, analyte recovery, and preparative capabilities remain. This will be addressed by future technological improvements that will ensure continuing new applications of ACE in the years to come.

Interconverting conformations of variants of the human amyloidogenic protein beta2-microglobulin quantitatively characterized by dynamic capillary electrophoresis and computer simulation.

Capillary electrophoretic separation profiles of cleaved variants of beta2-microglobulin (beta2m) reflect the conformational equilibria existing in solutions of these proteins. The characterization of these equilibria is of interest since beta2m is responsible for amyloid formation in dialysis-related amyloidosis and thus is able to attain alternative conformations that lead to irreversible aggregation and precipitation. In this study, we quantitate the increased conformational instability of cleaved beta2m by extracting rate constants and activation energies by simulating the experimental data using a unified theory for dynamic chromatography and dynamic electrophoresis. The results are correlated with the outcome of independent experiments based on mass spectrometric measurement of H/D exchange. This study illustrates that dynamic capillary electrophoresis is suitable for the investigation of the interconversion of protein conformations of amyloidogenic molecules and is not only restricted to ideal model compounds.

Sample handling for mass spectrometric proteomic investigations of human sera.

Proteomic investigations of sera are potentially of value for diagnosis, prognosis, choice of therapy, and disease activity assessment by virtue of discovering new biomarkers and biomarker patterns. Much debate focuses on the biological relevance and the need for identification of such biomarkers while less effort has been invested in devising standard procedures for sample preparation and storage in relation to model building based on complex sets of mass spectrometric (MS) data. Thus, development of standardized methods for collection and storage of patient samples together with standards for transportation and handling of samples are needed. This requires knowledge about how sample processing affects MS-based proteome analyses and thereby how nonbiological biased classification errors are avoided. In this study, we characterize the effects of sample handling, including clotting conditions, storage temperature, storage time, and freeze/thaw cycles, on MS-based proteomics of human serum by using principal components analysis, support vector machine learning, and clustering methods based on genetic algorithms as class modeling and prediction methods. Using spiking to artificially create differentiable sample groups, this integrated approach yields data that--even when working with sample groups that differ more than may be expected in biological studies--clearly demonstrate the need for comparable sampling conditions for samples used for modeling and for the samples that are going into the test set group. Also, the study emphasizes the difference between class prediction and class comparison studies as well as the advantages and disadvantages of different modeling methods.

Determination of octanol-water partition coefficients for carbonate esters and other small organic molecules by microemulsion electrokinetic chromatography.

Microemulsion electrokinetic chromatography (MEEKC) was assessed as a tool for determination of octanol-water partition coefficients using 34 solutes encompassing 8 carbonate esters. It was confirmed that microemulsions containing 1.44-2.88% w/w SDS, 6.49% w/w 1-butanol, and 0.82% w/w n-heptane constitute a good model of octanol-water partitioning in the pH range of 1.4-7.4. Use of the migration index concept led to improved repeatability of the MEEKC method compared to the use of retention factors. Using a dynamical coating, a high electroosmotic flow at pH 1.4 and 4.75 was achieved expanding the practical pH working range of the MEEKC system. The correlation obtained between the migration index and log P was unaffected by pH indicating that the properties of the microemulsion droplets and, thus, partitioning are independent of pH. No evidence for congeneric behavior was found for the sample set comprising solutes with different hydrogen bonding properties suggesting that simple reference compounds can be used as calibrators. Lipophilicity estimates for the series of carbonate esters were obtained. The increase in lipophilicity with chain length was smaller than expected from the Hansch substituent constant, pi.

Effect of dextran as a run buffer additive in drug-protein binding studies using capillary electrophoresis frontal analysis.

The study of drug-protein interactions by capillary electrophoresis frontal analysis requires establishment of a sufficient mobility difference between the mobility of the ligand and protein. The potential utility of dextran as a run buffer additive to manipulate the electrophoretic mobilities of low molecular weight ligands and protein in capillary electrophoresis frontal analysis binding studies was assessed. It was demonstrated that dextran was effective in improving the separation between the ligands warfarin and flurbiprofen and human serum albumin. Separation of ligand and protein increased with the concentration of added dextran (0-7.5% (w/w)), while molecular weight of the additive (70,000-2,000,000) only had a minor effect. The effect of dextran addition on viscosity and electrophoretic and electroosmotic mobilites was systematically studied. Optimal frontal analysis settings were a compromise between achieving satisfactory separation and acceptable analysis times without loss of plateau peak conditions. No effect of dextran upon the drug-human serum albumin interactions could be detected for the model ligands. Introduction of dextran into the electrophoresis buffer expands the applicability of capillary electrophoresis frontal analysis in drug research to binding interactions between proteins and low molecular weight ligands possessing similar electrophoretic mobilities.

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions.

Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug-human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV-detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.