1.5 Proteinanalytik
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- 1 Analytische Chemie; Referenzmaterialien (24)
- 1.5 Proteinanalytik (24)
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- 1.1 Anorganische Spurenanalytik (2)
- 1.9 Chemische und optische Sensorik (2)
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Eingeladener Vortrag
- nein (2)
Das Ziel der Arbeit war die Entwicklung einer magnetischen Anreicherungsmethode für eine Bead-basierte Substanzbibliothek zur Identifizierung von Proteinbindern. Im Ramen des Forschungsbelegs wurde bereits eine Anreicherungsmethode für ein Modellsystem einer Peptidbibliothek entwickelt. Das Modellsystem bestand dabei aus zwei verschiedenen Peptiden. Als Positivkontrolle wurde das FLAG-Peptid gewählt, welches selektiv gegen den Anti-FLAG-Antikörper bindet, und ein weiteres Peptid wurde als Negativkontrolle gewählt. Anhand dieses Modellsystems konnte die zuvor entwickelte Anreicherungsmethode von 20.000 auf 1.000.000 Beads vergrößert werden. Hierbei wurde ein Anreicherungsfaktor von 818 und eine gute Wiederfindungsrate von 82% erreicht.
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.
Hepcidin-25 concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state-of-the-art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95 ± 0.11 nmol/L) and middle concentration (3.75 ± 0.17 nmol/L) calibrators of the sRM. Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin-25.
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.
Synthetic peptide pools are used in antigen-specific T-cell assays, which are an important part in vaccine and immunotherapeutic clinical trials. As the analytical characterization is challenging due to the similarity of the single peptides or is expensive due to isotope labeled standards, usually only a pre-characterization of the single peptides is performed. However, a regular quality control of the peptide mix would be highly desirable. Therefore, a cost-efficient high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method for quality control of a model peptide pool is developed.
Peptides were synthesized using peptides&elephants proprietary libraries of individual peptides (LIPS) technology and purified by reversed-phase chromatography to > 90% each. The lyophilized single peptides were combined to a model peptide pool and analyzed by reversed-phase high-performance capillary liquid chromatography coupled to an orbitrap mass spectrometer. Separation was performed on a capillary reversed phase column (2 μm, ID x L 300 μm x 150 mm) with a linear gradient of acetonitrile + 0,05% trifluoroacetic acid.
For the separation of a model peptide pool the additive, additive concentration, the gradient elution and the temperature were optimized. Different quantification approaches were tested. Identification was performed by high resolution mass spectrometry in which extracted ion chromatograms (XIC) were used to confirm exact masses.
In line with the development of a new cost-efficient quality control method for the separation and identification of complex synthetic peptide pools, varied HPLC parameters highlighted their influence on chromatographic resolution and peptides were identified with high mass accuracy.
Spec2Seq
(2018)
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.