1.5 Proteinanalytik
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- Magnetic resonance imaging (13)
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Organisationseinheit der BAM
- 1 Analytische Chemie; Referenzmaterialien (125)
- 1.5 Proteinanalytik (125)
- 1.9 Chemische und optische Sensorik (16)
- 1.8 Umweltanalytik (13)
- 6 Materialchemie (11)
- 1.1 Anorganische Spurenanalytik (10)
- 6.3 Strukturanalytik (10)
- 8 Zerstörungsfreie Prüfung (7)
- 8.1 Sensorik, mess- und prüftechnische Verfahren (7)
- 1.7 Organische Spuren- und Lebensmittelanalytik (5)
Eingeladener Vortrag
- nein (9)
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.
After optimizing the injection mode, the gradient elution, the temperature and the additives a model peptide pool was separated. The extracted ion chromatogram (XIC) was studied to confirm the exact masses.
By combination of capillary HPLC and HRMS a new cost-efficient quality control method could be developed for the separation and identification of complex synthetic peptide pools.
In this work, a cost-effective optofluidic system is proposed and preliminary experimental results are presented. A microfluidic channel monolithically integrated into a photonic integrated circuit technology is used in conjunction with a cyclic olefin copolymer (COC) substrate to provide fluidic in- and output ports. We report on initial experimental results as well as on the simple and cost-effective fabrication of this optofluidic system by means of micro-milling.
Remote sensing of hazardous substances is a key task that can be achieved with the help of remotely operated platforms equipped with specific sensors. A huge variety of methods and used vehicles have been developed for different purposes in recent years. The term smart dust refers to a science fiction novel and develop shortly after into a research proposal at UC Berkley funded by DARPA. Subsequently, the topic gained attraction but was overall considered as to complex for the technologies available at that time. In the launched passive smart dust project, we shift to a simple “chemical intelligent” passive sensor particle on the ground combined a read-out active sensor attached to an Unmanned Aerial Vehicle (UAV). The reactive particle surface can be preadjusted in the lab for exact desired properties regarding certain reactions to hazardous substances.
Moreover, the aimed interaction with the active sensor can be modified. Planed applications allow for different materials e.g., for short time measurement, being ecologically degradable, or weather stable for long time monitoring.
The fundamental properties of a polymeric material are ultimately governed by its structure, which mainly relies on monomercomposition and connection, topology, chain length, and polydispersity. Thus far, these structural characteristics are typicallyset ex situ by the specific polymerization procedure, eventually limiting the future design space for the creation of moresophisticated polymers. Herein, we report on a single photoswitchable catalyst system, which enables in situ remote controlover the ring-opening polymerization of l-lactide and further allows regulation of the incorporation of trimethylene carbonateand δ -valerolactone monomers in copolymerizations. By implementing a phenol moiety into a diarylethene-type structure,we exploit light-induced keto–enol tautomerism to switch the hydrogen-bonding-mediated monomer activation reversiblyON and OFF. This general and versatile principle allows for exquisite external modulation of ground-state catalysis of a livingpolymerization process in a closed system by ultraviolet and visible light and should thereby facilitate the generation of newpolymer structures.
ABID
(2022)
In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.
ADAMTS4-specific MR-probe to assess aortic aneurysms in vivo using synthetic peptide libraries
(2022)
The incidence of abdominal aortic aneurysms (AAAs) has substantially increased during the last 20 years and their rupture remains the third most common cause of sudden death in the cardiovascular field after myocardial infarction and stroke. The only established clinical parameter to assess AAAs is based on the aneurysm size. Novel biomarkers are needed to improve the assessment of the risk of rupture. ADAMTS4 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 4) is a strongly upregulated proteoglycan cleaving enzyme in the unstable course of AAAs. In the screening of a one-bead-one-compound library against ADAMTS4, a low-molecular-weight cyclic peptide is discovered with favorable properties for in vivo molecular magnetic resonance imaging applications. After identification and characterization, it’s potential is evaluated in an AAA mouse model. The ADAMTS4-specific probe enables the in vivo imaging-based prediction of aneurysm expansion and rupture.
Therapeutische Antikörper sind innerhalb weniger Jahre zur wichtigsten pharmazeutischen Produktklasse aufgestiegen. Für 2023 werden weltweite Umsätze von über 200 Milliarden USD erwartet. Auch diagnostische Antikörper sind mittlerweile unverzichtbare Produkte, auf deren Basis zahllose Immunoassays und andere Schnelltests entwickelt wurden. Neben der bereits sehr aufwendigen Herstellung von Antikörpern ist deren Aufreinigung aus komplexen Zellkulturmedien oder Blutseren und -plasmen zu einem Engpass in der Produktion und Nutzung dieser komplexen Proteine geworden. Schnelle und einfache Reinigungsmethoden für Antikörper sind daher sehr gefragt.
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.
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.
The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often, critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence, a risky venture. We think that it is crucial to improve the screening process to eliminate most of the critical deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high-throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and performance of simultaneous competition experiments. The latter can also be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones, and blank supernatant containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media is used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones.