1.5 Proteinanalytik
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Communities worldwide face significant threats from Explosive Remnants of War (ERW), which endanger lives and restrict land usage. From forest fires due to ERWs or in ERW-contaminated areas (e.g., in Jüterbog, Germany) to broader global challenges (e.g., the Ukrainian conflict), the need for efficient detection and removal of these remnants, especially for humanitarian demining, is paramount. Traditional methods, like manual demining, have severe limitations in safety and efficiency. Here, we introduce an innovative solution to these challenges: “Chemosensing Smart Dust.” This technology uses chemoselective dyes that change their fluorescence properties when exposed to explosives like 2,4,6-trinitrotoluene (TNT). Fluorescence-based detection offers superior sensitivity, reduced likelihood of false positives, and enhanced accuracy of explosive detection. Drones, equipped with excitation lasers or LEDs, deploy the Chemosensing Smart Dust over areas of interest and actively detect the fluorescence changes using high-resolution cameras, offering a rapid, safe, and adaptable detection method. Beyond demining, this innovative approach has potential applications in monitoring polluted areas, homeland security, and emergency response.
Screening against ADAMTS4 reveals a specific peptide, which was turned into an MRI probe. The aneurysm in a mouse modal was visualized via MRI. A differentiation between stable and unstable aneurysm in an early state was performed. Using the probe as tool for an easy and non-invasive rupture assessment is possible.
Nonporous corundum powder was functionalized covalently with protein binders to isolate and enrich specific proteins from complex matrices.The common crosslinker glutaraldehyde was compared with a hyperbranched polyglycerol (PG) of around 10 kDa. The latter was oxidized with periodate to generate aldehyde groups that can covalently react with the amines of the surface and the amino groups from the protein via a reductive amination process. The amount of bound protein was quantified via aromatic amino acid analysis (AAAA). As a proof of concept, IgG was extracted with protein A from crude human plasma. The advantages of corundum include the very low price, extremely high physical and chemical stability, pressure resistance, favorable binding kinetics, convenient handling, and flexible application.
High demand for remote sensing of hazardous substances. Possible solution: Use of distributed, low cost, and environmentally safe particles as passive sensors that can be read out remotely
Chemical intelligence on the particle surface can be easily modified Particles enable optically quantifiable response and inference of target substances (also no maintenance or power supply required
Cardiovascular diseases are the number one cause of death worldwide and responsible for 3.9 million deaths in Europe and over 1.8 million deaths in the European Union (2017) [1]. Cardiac Troponin I (cTnI) is part of a triple protein complex and the most important biomarker for acute events such as heart attacks. Once a heart attack occurs, the blood stream within the heart is interrupted and further oxygen supply cannot be maintained. As a result, heart cells undergo apoptosis and their proteins will be released into the blood stream. Within the project CardioMet of the European Metrology Program of Research and Innovation (EMPIR) a biosensor for online-monitoring of acute myocardial infarction by detecting cTnI is being developed.
Antibodies are the most used biomolecules in analytical research. Nevertheless, the sequence and structure information of antibodies is often limited, since manufacturers keep them secret or suppliers sell them under different names. This can make it difficult to reproduce even basic experiments performed in publications as the antibodies used might not be identifiable. To overcome these problems, we developed a simple and cheap method for antibody identification by MALDI-TOF-MS fingerprinting. This technique was used to generate a library of antibody fingerprints, which enables the identification and comparison of antibodies in short time.
The antibody A.1.1.1 was labeled and found to be very sensitive and highly selective for TNT. A novel monolithic affinity column was coated with a Trinitroaniline (TNA)-BSA affinity conjugate and a custom laser induced fluorescence detector were built to allow for continuous and sensitive detection. The affinity column combined with the high sensitivity detector resulted in a limit of detection of approx. 100 pM TNT or 20 ppt TNT for offline detection and was able to detect picogram amounts within three minutes.
The mechanism of this system is based on kinetic competition. This biosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. If the explosive 2,4,6-trinitrotoluene (TNT) is introduced some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein. The fluorescence is detected by highly sensitive laser-induced fluorescence with a conventional CMOS camera. The system achieved limits of detection of approx.1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of TNT. The total assay time is less than 8 minutes. A cross-reactivity test with 5000 pM solutions of pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) showed no cross reactivity.
The urgent necessity to carry out reliable and relevant analytical measurements directly at a point-of-need is one of the current drivers for the development of miniaturised analytical systems, quick tests and wearables. Despite their simplicity, this type of tests must guarantee analytical relevance and reliability like laboratory-based analysis, e.g., in terms of sensitivity, selectivity, immunity against false positives and false negatives as well as robustness and repeatability. Keeping in mind the high sensitivity offered by gated indicator-releasing micro- and nanoparticles due to their inherent features of signal amplification, we performed several optimisations to develop a potential biosensor platform for use in rapid tests. Conceptually, these gated materials are closely related to drug delivery systems, consisting of high porous materials usually closed with macromolecular “caps” and loaded with indicator molecules that are released in presence of a target analyte. However, the key difference between the two types of functional materials is that many drug delivery systems should deliver their cargo over a longer period, often many hours, whereas the gated materials prepared for sensing should show fast release kinetics, on the order of <5 min.
With the aim to optimise and adapt gated materials for sensing purposes, we prepared in this work several antibody-gated materials for small-molecule sensing. The materials consisted of porous silica particles containing indicator molecules in the pores and certain hapten molecules grafted to the particle surface close to the pore openings. The pores were then capped with antibodies binding to these haptens, thus inhibiting the escape of the indicators from inside of the pores. In presence of the corresponding analyte, the antibody is displaced from the surface of the material, allowing the escape of the indicators. This allows the detection of the analyte indirectly through an inherent signal amplification. In this work, the insecticide permethrin, a type-I pyrethroid, was selected as target model, because type-I pyrethroids play an important role in airplane disinfection. A first in-depth study of the various chemical tuning options of such antibody gated systems was performed. Different mesoporous silica supports, different functionalisation routes and different loading sequences were assessed. The materials’ performances were evaluated by studying their temporal response behaviour and detection sensitivity, including the tightness of pore closure (through the amount of blank release in absence of analyte) and the release kinetics. Our results indicate that the better the paratope-accommodating Fab region of the antibody “cap” fits into the host material’s pore openings, the better the closing/opening mechanism can be controlled. Because such materials can be used in various different formats from suspension assays[1] via microfluidic chips[2] to test strip-based lateral flow assays,[3] such materials present a powerful analytical particle platform for the sensitive analytics and diagnostics outside of a laboratory, realising sensitivities down to the µg kg–1 range in less analysis times of less than 5 min as we have recently demonstrated.[4]
In this study, we developed a new immunoprecipitation method based on protein-coated nanodiamonds. Performing SDS-PAGE and using the SDS-PAGE buffer as an eluent showed that CER could be successfully enriched from human serum. Based on the copper determination with ICP-MS, the amount of bound CER on the nanodiamonds can be calculated. We could show the fulfilled mass balance of bound CER and CER in the supernatant after incubation with a known amount of CER. For isotope ratio analysis this method can be applied to compare ratios of the total copper content in human serum to copper ratios from CER enrichment.
An affinity column, which removed up to 99 % of high affinity Anti-TNT antibody at high antibody concentrations, was manufactured based on a monolithic glass core and a trinitroaniline-BSA conjugate. To detect the label Dy654 in the nM range an epi-fluorescence microscope setup with a CMOS camera was established to serve as online fluorescence detector with multiplexing capabilities.
Screening of one-bead-one-peptide libraries is a powerful analytical tool for the identification of protein ligands. However, the traditional peptide screening procedure involves tedious steps such as manual selection, sequencing, and characterization. We present a high-throughput “all in one chip” system, allowing the screening of a high number of resin beads in short time. Here, beads of a combinatorial one-bead one compound peptide library are immobilized on an in-house produced chip, on which every bead has a well-defined position. The chip is then incubated with a fluorophore-labeled protein, identifying suitable peptides by a high-resolution fluorescence scan. The screening is followed by MALDI-MS experiments directly on the respective glass chip. To circumvent the need for peptide fragmentation normally used for peptide de novo sequencing, which can result in incomplete sequence information, an approach based on ladder sequencing has been used. This allows the peptide sequence identification by fragmentation-free MS with almost 100 % accuracy. For this purpose, a software tool was developed automatically translating MALDI-MS spectra into the corresponding peptide sequences.
Spheriplex multiplexed immunoassays: A practical tool for environmental, food and bioanalysis
(2019)
Clinical, environmental, and food analysis, require efficient and accurate quantitative analysis. Immunoassays take advantage of highly selective and affine binding of an antibody towards an antigen, being easy-to-use and allowing for high sample throughput. However, common immunoassays, e. g. ELISA are not capable of multiplexed analysis, thus limiting possible applications. On the other hand, multi-analyte methods, e.g. liquid chromatographytandem mass spectrometry requires expensive equipment, trained analysts and the time of analysis usually limits sample throughput. This bottleneck can be overcome combining the suspension array technology, where particles are encoded to allow multiplexed detection, with fluorescence immunoassays to create suspension array fluorescence immunoassays (SAFIA).
Polystyrene core/silica shell microparticles serve as platform for SAFIA. While an encoding dye is introduced in the polystyrene core, the silica shell is used for immobilization: For competitive hapten immunoassays small molecules are immobilized on the surface, serving as a competitive binding site for selective antibodies. For quantification of proteins, antibodies can be immobilized on the surface and the sandwich immunoassay format can be employed. All mentioned immunoassays can be executed wash-free and highly parallelized, allowing up to 500 determinations within three hours. In contrast to ELISA, short incubation times, a wash-free mix-and-read procedure and automated flow-cytometric read-out makes SAFIA applicable for even non-trained personal.
SAFIA showed excellent performance in studies on the origin and fate of drug residues in waste water, which is important to assess clearance rates of wastewater treatment plants. Furthermore, SAFIA can be employed in environmental screenings, detecting pollution of natural river waters by tracking anthropogenic marker substances. It was successfully employed in clinical and food analysis applications, e.g. the quantification of the anti-inflammatory drug diclofenac in breast milk or the quantification of caffeine in beverages. Due to multiplexing capacities, SAFIA can significantly improve biotechnological processes, as in the screening of hybridoma cells for selective and efficient antibody production. For small molecule analytes, limits of detection down to 4 ng/L and for proteins below 25 ng/mL (IgG) were observed, which makes SAFIA applicable to all addressed analytical issues. Moreover, in the analysis of real-world samples, SAFIA shows higher accuracy in contrast to ELISA, indicating higher matrix stability and thus higher robustness in analysis.
Type-I pyrethroids are frequently used for disinfection purposes against insects such as adult mosquitoes, or diseases carried by insects, like Malaria or Zika in cabins of airplanes on long-distance flights especially from tropical destinations. This treatment is mandatory at various airports but compliance with the rules is difficult to test for. Moreover, if improperly used, these compounds can entail negative health effects for crews and passengers.
The detection of the pyrethroids will be achieved thanks to an antibody-gated indicator delivery system (gAID) utilizing monoclonal antibodies and hybrid sensory nanoparticles. After the interaction of the pyrethroid with the gAID, the liberated indicator (dye) will be detected. Since only few analyte molecules are necessary for pore opening yet release a large number of dyes, the system shows intrinsic signal amplification.
The device system to be developed has to be so simple that chemically untrained personnel, such as ground or cabin crew, can use it and obtain a result in a reasonably short period of time, e.g., ≤5 min. The need for high accuracy and sufficient sensitivity, established at 0.001 g m–2, is a critical requirement and imposes another significant challenge since this value is beyond current LFTs reported in the literature for pesticide detection to date.
In order to achieve the selectivity and sensitivity required by the test itself, and to avoid cross reactivity with other type I pyrethroids, the production of a monoclonal antibody for both Permethrin and Phenontrin is necessary. The synthesis of the two hapten molecules and the subsequent immunization with different immunogens represent the first goal of the work.
Comparison of 1H chemical shifts and TOCSY data in the presence and absence of Ni2+ demonstrates that the metal binds at the N-terminus of the hepcidin-25. Chemical shift changes due to metal complexation decrease further away from the metal binding site, with the smallest effect at phenylalanine [Phe-4], proline [Pro-5] and isoleucine [Ile-6].
Hepcidin-25 regulates iron homeostasis in response to inflammation, erythropoietic demand and iron stores. The liver synthesizes three types of hepcidin, only hepcidin-25 indirectly regulates and coordinates use and storage of iron. Hepcidin-25 contains a well defined β-sheet and β-hairpin loop stabilized by four disulfide bonds. The N-terminus which plays a crucial role in the biological activity of hepcidin-25 was found to be disordered. The ATCUN motif sequence is present at the N-terminus (Asp-Thr-His). The ATCUN motif (H2N-X-X-His) binds Cu2+ and Ni2+ with high affinity and always contains histidine in its sequence. NMR provides an ideal tool to determine hepcidin-25’s threedimensional(3D) structure taking into account the Cu2+ and Ni2+ binding capacity of hepcidin’s ATCUN motif.
Expression, purification and characterization of the recombinant cysteine-rich biomarker Hepcidin-25
(2018)
Hepcidin regulates iron homeostasis in response to inflammation, erythropoietic demand, and iron stores. The native state of hepcidin-25 is an attractive target for the development of a reliable analytical tool that can quantify the hepcidin concentration in biological samples and reveal iron metabolic disorders. Therefore, a selective immunoassay would have to discriminate between different types of hepcidin and quantify only hepcidin-25’s concentration. The peptide contains a well-defined β-sheets and a β-hairpin loop stabilized by four disulfide bonds. Recently, it was shown that hepcidin-25 contains an ATCUN motif at its N-terminus. This motif is known to have high affinity towards Cu2+ and Ni2+.
One of the aims of this study is to determine the three-dimensional (3D) structure of metal-bound hepcidin-25. Here, we present an optimized procedure for preparing natively folded hepcidin 25 (~2.80 kDa) and structural analysis of metal binding to hepcidin-25. Hepcidin was expressed as a His6-SUMO-hepcidin-25 fusion protein (~16.20 kDa) in Escherichia coli, Origami B strains, and purified as a soluble recombinant protein in three steps. After purification based on the nickel affinity chromatography, the purified His6-SUMO-hepcidin 25 fusion protein was cleaved by the SUMO-specific ULP1 protease. The liberated hepcidin 25 was further purified on a Superdex 30 16/600 column and folded in the last step of purification in the presence of glutathione. Freshly expressed hepcidin was kept in its reduced form to prevent misfolding and allow for efficient removal of the SUMO tag. The presence of natively folded hepcidin 25 after RP-HPLC was confirmed by ESI-MS and NMR spectroscopy. Based on published chemical shifts, we achieved a nearly complete assignment of the labeled and unlabeled hepcidin-25 at pH=3. Comparison of 1H chemical shifts and TOCSY spectra at pH=7 in the presence and absence of Ni2+ demonstrates that the metal binds at the N-terminus of hepcidin 25. Chemical shift changes due to metal complexation decrease further away from the metal binding site.
Laser-induced plasma (LIP) has drawn significant amount of attentions in the past decades, particular in elemental analyses for solid or liquid samples. Through proper focusing of the highly energetic laser beam, the plasma can also be ignited in the ambient air, where airborne analytes can be ionized. Such an effect enabled the use of airborne LIP as an ambient ionization source for mass spectrometric analyses. In contrast to other ambient desorption/ionization sources, airborne LIP does not require a specific discharge medium or expensive gas stream. Meanwhile, the airborne LIP produces reagent ion species for both proton-transfer and charge-transfer reactions in addition to the vacuum ultraviolent photons that are capable of promoting single photon ionization, which can be utilized to ionize polar and non-polar analytes. In order to gauge the analytical performance of airborne LIP, it is critical to understand the undergoing chemistry and physics during and after the plasma formation.
Due to the ambient nature of airborne LIP, the variations of air composition and flow strongly affect the plasma behaviors. Preliminary result suggested the addition of a laminar flow of nitrogen gas favored the formation of protonated species (MH+) against the molecular ones (M+). Although the gas addition approach cannot fully tune the ionization process towards the specific production of pseudo-molecular species versus molecular ones, the alternation of molecular ion formation can be used for analyte recognitions through post processing of the ion patterns. The pulsed character of the used lasers makes the reagent ion equilibrium both transient- and highly fluid-dynamically controlled. The acoustic shock-waves induced by the airborne LIP get affected by an applied gas streams towards the plasma center, influencing the molecular-ion and ion-ion interactions in the near proximity of the plasma.
To understand the airborne LIP formation, the temporally and spatially resolved optical emission spectra were recorded. The results will be correlated to time-resolved mass-spectrometric investigations of the ion profile during different stages of the plasma formation. As one example, the formation of pyrylium ion originating from aromatic compounds will be highlighted.
Phage display is used to find specific target binding peptides for polypropylene (PP) surfaces. PP is one of the most commonly used plastics in the world. Millions of tons are produced every year. PP binders are of particular interest because so far gluing or printing on PP is challenging due to its low surface energy. A phage display protocol for PP was developed followed by Next Generation DNA Sequencing of the whole phage library. Data analysis of millions of sequences yields promising peptide candidates which were synthesized as PEG conjugates. Fluorescence-based adsorption-elution-experiments show high adsorption on PP for several sequences.
Small organic molecule drugs are one of the key classes, taking increasingly important roles in modern drug development strategies. With the focus on small molecule drugs, difficulties originate frequently from a pronounced lipophilic character, resulting in poor water solubility, low bioavailability and unfavored pharmacokinetics. Recently, peptide-poly(ethylene glycol) conjugates (peptide-PEG conjugates) were described as precisely tunable platforms to solubilize a broad scope of fluorescent or non-fluorescent small organic molecules [1-2]. Selection of drug hosting peptides was achieved by combinatorial means, which can further be extended by implementation of a drug release screening step.
One-bead-one-compound peptide libraries are powerful tools to select high affinity binders. However, the selection of positive hits from the peptide libraries remains tedious as it occurs by handpicking, strongly limiting the pool of investigated beads. Here we report our recent results on improving the analytical platform, using automated fluorescence scanning and MALDI-ToF-ToF MS/MS imaging to screen larger sets of beads, broadening the statistical base and unraveling more precisely suitable peptides. The screening puts special emphasis on loading capacities and drug-release of transporters by performing additional washing steps in different media (cf. Fig. 1). Peptides representing strong, medium, weak releaser were chosen for further analysis and synthesized as peptide-polymer transporters. Release was analyzed by fluorescence anisotropy and fluorescence correlation spectroscopy, due to the fluorescent characteristics of the drug.
Solubilization studies confirmed sufficient loading capacities for a potential anti-Alzheimer disease drug of three transporter molecules representing strong/weak drug releaser, reaching solubilization of up to 1:3.4 (µmol drug/µmol conjugate). Fluorescence anisotropy and fluorescence correlation spectroscopy of the drug-loaded transporter showed significant differences in drug releasing properties, confirming the screening process.
Hepcidin-25 has attracted much attention ever since its discovery in 2001. It is widely recognized that this peptide hormone plays a major role in the regulation of iron levels in mammals and can reveal important clinical information about several iron-related disorders. However, the development of a reliable assay to quantify hepcidin proved to be problematic and serum hepcidin-25 concentrations determined by various assays differ substantially. Challenges arise in the MS analysis of hepcidin due to the “sticky” character of the peptide and the lack of suitable standards.
With the aim to tackle the current difficulties in hepcidin quantification and improve the status of this promising biomarker in the clinical field, we developed a rapid and robust analytical strategy for the quantification of hepcidin-25 in human samples based on HPLC-MS/MS (QqQ) as a reference method candidate to be implemented in routine laboratories. The novelty of the method is the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces. Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions where the use of acidic mobile phases was compared with a novel approach involving solvents at high pH containing 0.1% of ammonia. Both methods were carefully validated and applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with very good correlation of the results.
Screening of one-bead-one-peptide libraries is a useful procedure for the identification of protein ligands. The demand for binders with high affinity and high specificity towards various target proteins has surged in the biomedical field in the recent years. The traditional peptide screening procedure involves tedious steps as selection, sequencing, and characterization. Herein, we developed a high-throughput – “all in one chip” system to avoid time-consuming separation steps.
Multidimensional high throughput methods for the analysis of particle-based peptide libraries
(2018)
Screening of one-bead-one-peptide libraries is a useful procedure for the identification of protein ligands. The demand for binders with high affinity and high specificity towards various target proteins has surged in the biomedical field in the recent years. The 1 traditional peptide screening procedure involves tedious steps as selection, sequencing, and characterization. Herein, we developed a high-throughput – “all in one chip” system to avoid time-consuming separation steps.