1.5 Proteinanalytik
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- 2018 (24) (entfernen)
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- Copper (6)
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- Chromatography (3)
- Iron disorders (3)
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- Nickel (3)
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- 1 Analytische Chemie; Referenzmaterialien (24)
- 1.5 Proteinanalytik (24)
- 1.8 Umweltanalytik (3)
- 1.1 Anorganische Spurenanalytik (2)
- 1.9 Chemische und optische Sensorik (2)
- 1.3 Instrumentelle Analytik (1)
- 6 Materialchemie (1)
- 6.3 Strukturanalytik (1)
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Eingeladener Vortrag
- nein (2)
The quality of research antibodies is an issue for decades. Although several papers have been published to improve the situation, their impact seems to be limited. This publication makes the effort to simplify the description of validation criteria in a way that the occasional antibody user is able to assess the validation level of an immunochemical reagent. A simple, 1-page checklist is supplied for the practical application of these criteria.
In dieser Arbeit wurden drei verschiedene Nanomaterialien auf ihre Bindungsfähigkeit zu Proteinen untersucht. Zu Beginn standen dabei die Herstellung stabiler Dispersionen der einzelnen Nanopartikel und die Stabilität der gebildeten Konjugate im Vordergrund. Der Nachweis einer erfolgreichen Konjugatbildung, sprich der Beschichtung von Nanopartikel mit Proteinen, wurde sowohl qualitativ mittels DLS-Messungen als auch über quantitative Protein-Bestimmungen erbracht. Für die Quantifizierung konnten verschiedene Methoden eingesetzt werden. Neben der klassischen Vorgehensweise, welche indirekt über die Quantifizierung von ungebundenem Protein im Überstand erfolgt, konnten ihm Rahmen dieser Arbeit verschiedene direkte Bestimmungsmethoden entwickelt werden. So wurden mittels kolorimetrischer Tests, wie dem BCA-Assay und dem Bradford-Assay, Nanodiamantdispersionen mit Hilfe einer Korrekturwellenlänge vermessen und quantifiziert. Ebenso zum Einsatz kam die Methode der Aminosäureanalytik, welche aufgrund ihrer guten Rückführbarkeit auf Aminosäurestandards Ergebnisse mit hoher Richtigkeit generieren kann und ebenso die Detektion kleiner Proteinmengen möglich macht.
Nach den erfolgten quantitativen Betrachtungen wurden die Protein-beschichteten Nanopartikel auf ihre Anwendbarkeit als Analoga von Virus-like Particles (VLP) bei einer Immunisierung zur Gewinnung von polyklonalen Antikörpern gegen humanes Ceruloplasmin in Kaninchen überprüft. Es konnte mittels ELISA gezeigt werden, dass die Konjugate erfolgreich für die Herstellung von Antikörpern eingesetzt werden können und im zeitlichen Verlauf einer Immunisierung eine Steigerung des Antikörper-Titers zu erreichen ist.
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.
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.
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].
Spec2Seq
(2018)
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.
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.
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.