Dokument-ID Dokumenttyp Autoren/innen Persönliche Herausgeber/innen Haupttitel Abstract Auflage Verlagsort Verlag Herausgeber (Institution) Erscheinungsjahr Titel des übergeordneten Werkes Jahrgang/Band ISBN Veranstaltung Veranstaltungsort Beginndatum der Veranstaltung Enddatum der Veranstaltung Ausgabe/Heft Erste Seite Letzte Seite URN DOI Lizenz Datum der Freischaltung OPUS4-56452 misc Tchipilov, Teodor; Meyer, Klas; Weller, Michael G. Quantitative 1H Nuclear Magnetic Resonance (qNMR) of Aromatic Amino Acids for Protein Quantification qNMR is a valuable technique for metrological studies due to the uniformity of its signal response for all chemical species of an isotope of interest, which enables compound-independent calibration. However, protein quantification remained challenging as large molecules produce wide, low-intensity signals that reduce the already low sensitivity. Combining qNMR with the hydrolysis of protein samples into amino acids circumvents many of these issues and facilitates the use of NMR spectroscopy for absolute protein and peptide quantification.In this work, different conditions have been tested for quantifying aromatic amino acids and proteins. First, we examined the pH-based signal shifts in the aromatic region. The preferable pH depends on the selection of the amino acids for quantification and which internal standard substance should be used to avoid peak overlap. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, have been applied as internal standards. The quantification of amino acids from an amino acid standard, as well as from a certified reference material (bovine serum albumin), was performed. Using the first two suggested internal standards, recovery was ~ 97 % for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98 ± 2 and 88 ± 4 %, respectively, at a protein concentration of 16 g/L or 250 µM. Basel MDPI 2022 Preprints 1 13 urn:nbn:de:kobv:b43-564520 10.20944/preprints202211.0569.v1 https://creativecommons.org/licenses/by/4.0/deed.de 2022-12-05 OPUS4-54536 misc Tchipilov, Teodor; Raysyan, Anna; Weller, Michael G. Methods for the quantification of particle-bound protein - Application to reagents for lateral-flow immunoassays (LFIA) Protein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such as Bradford, bicinchoninic acid (BCA), as well as AAAA of the supernatant. For both particle types, these indirect quantification techniques show a protein overestimation of up to 1700% compared to the direct AAAA measurements. In addition, protein coating on latex particles was performed both passively through adsorption and covalently through EDC/sulfo-NHS chemistry. Our results showed no difference between the immobilization methodologies. This finding suggests that usual protein determination methods are no unambiguous proof of a covalent conjugation on particles or beads. Basel MDPI 2022 Preprints 1 8 urn:nbn:de:kobv:b43-545365 10.20944/preprints202203.0332.v1 https://creativecommons.org/licenses/by/4.0/deed.de 2022-03-28 OPUS4-54518 misc Tscheuschner, Georg; Kaiser, Melanie N.; Lisec, Jan; Beslic, D.; Muth, Thilo; Krüger, M.; Mages, H.-W.; Dorner, B. G.; Knospe, J.; Schenk, J. A.; Sellrie, F.; Weller, Michael G. MALDI-TOF-MS-based identification of monoclonal murine anti-SARS-CoV-2 antibodies within one hour During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used thirty-five monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied onto the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 45 minutes and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0 (https://gets.shinyapps.io/ABID/). 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. Basel MDPI 2022 Preprints 1 24 urn:nbn:de:kobv:b43-545182 10.20944/preprints202203.0229.v1 https://creativecommons.org/licenses/by/4.0/deed.de 2022-03-23 OPUS4-53354 misc Iwert, C.; Stein, J.; Appelt, C.; Vogt, K.; Rainer, R. J.; Tummler, K.; Mühle, K.; Stanko, K.; Schumann, J.; Uebe, D.; Jürchott, K.; Lisec, Jan; Janek, K.; Gille, C.; Textoris-Taube, K.; Sai, S.; Petersen, A.; Kühl, A. A.; Klipp, E.; Meisel, C.; Sawitzki, B. TCAIM controls effector T cell generation by preventing Mitochondria-Endoplasmic Reticulum Contact Site-initiated Cholesterol Biosynthesis T cells need to adapt their cellular metabolism for effector cell differentiation. This relies on alterations in mitochondrial physiology. Which signals and molecules regulate those alterations remains unclear. We recently reported, that the mitochondrial protein TCAIM inhibits activation-induced changes in mitochondrial morphology and function and thus, CD effector T cell formation. Using conditional TCAIM knock-in (KI) and knockout (KO) mice, w now show that it also applies to CD8+ T cells and more importantly, delineate the molecular processes in mitochondria by which TCAIM controls effector cell differentiation. TCAIM KI resulted in reduced activation-induced HIF1α protein expression. Metabolomics and transcriptional data in combination with mathematical flux modeling revealed an impaired induction of anabolic pathways, especially of the mevalonate pathway and cholesterol biosynthesis in TCAIM KI CD8+ T cells. Addition of cholesterol completely rescued HIF1α protein expression, activation and proliferation of TCAIM KI CD8+ T cells. At the molecular level, TCAIM delayed activation-induced mitochondria-ER contact (MERC) formation by binding to MERC promoting proteins such as RMD3 and VDAC2. In summary, we demonstrate that TCAIM suppresses effector cell differentiation by inhibiting MERC formation, which induce HIF1α-mediated increase in cellular metabolism and cholesterol biosynthesis. Cold Spring Harbor Laboratory 2021 bioRxiv April 1 45 urn:nbn:de:kobv:b43-533543 10.1101/2021.04.20.440500 https://creativecommons.org/licenses/by-nc-nd/4.0/deed.de 2021-09-23 OPUS4-55514 misc Völzke, Jule L.; Hodjat Shamami, P.; Gawlitza, Kornelia; Feldmann, Ines; Zimathies, Annett; Weller, Michael G. High-purity corundum as support for affinity extractions from complex samples Nonporous corundum powder, known as an abrasive material in the industry, was functionalized covalently with protein binders to isolate and enrich specific proteins from complex matrices. The materials based on corundum were characterized by TEM, ESEM, BET, DLS, and zeta potential measurements. The strong Al-O-P bonds between the corundum surface and amino phosphonic acids are used to introduce functional groups for further conjugations. The common cross-linker glutaraldehyde was compared with a hyperbranched polyglycerol (PG) of around 10 kDa. The latter is 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). This work shows that oxidized polyglycerol can be used as an alternative to glutaraldehyde. With polyglycerol, more of the model protein bovine serum albumin (BSA) could be attached to the surface under the same conditions, and lower nonspecific binding (NSB) was observed. As a proof of concept, IgG was extracted with protein A from crude human plasma. The purity of the product was examined by SDS-PAGE. A binding capacity of 1.8 mg IgG per g of corundum powder was achieved. The advantages of corundum are the very low price, extremely high physical and chemical stability, pressure resistance, favorable binding kinetics, and flexible application. Basel MDPI 2022 Preprints 1 13 urn:nbn:de:kobv:b43-555142 10.20944/preprints202208.0004.v1 https://creativecommons.org/licenses/by/4.0/deed.de 2022-08-17 OPUS4-51162 misc Luco Colilles, Arnau Evaluation of two compact near-infrared spectrometers for the detection of microplastics in soil samples In the last years, the Process Analytical Technology (PAT) research field has become of great importance due to its connection with the industrial world. This industry related research, along with the evolutionary progress that technology has seen lately, has allowed that key analytical techniques in an industrial environment have undergone a radical development. One of these techniques has been the near-infrared spectroscopy (NIRS). This technique, currently common in "online" industrial analysis, has seen a remarkable revolution, especially since the introduction of the microelectromechanical systems technology (MEMS) in the spectroscopy field. Nowadays it is possible to find compact spectrometers no bigger than a wristwatch in the market. Although there is an unavoidable question: can these compact spectrometers actually compete against the traditional spectrometers? In this project different calibration parameters of two compact NIR spectrometers ("Spectral Engines Oy NIR-One Sensor NM2.0", with 1550 to 1950 nm range; and "NeoSpectra Si-ware", with 1300 to 2550 nm range) were evaluated, and the obtained results were compared with a reference spectrometer ("Bruker Optik GmbH Matrix-F", with 15000 to 4000 cm-1 range). In order to obtain the different calibration parameters, a sequence of quality performance tests were conducted. The results obtained after the different experiments carried out with both compact spectrometers prove that their performance is more than acceptable for routine analysis. Afterward, model samples of different microplastics in soil at different known concentration were analyzed with all three spectrometers. Chemometric models capable to identify and classify microplastics in soil were established. For this analysis five of the most used plastics worldwide were used: polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC). After the Principal component analysis (PCA), it can be seen that only the NIR-One NM2.0 is capable to differentiate all types of microplastics in soil at concentrations of 1-2 %, while the NeoSpectra Si-ware is unable to identify the PET sample. Barcelona Universitat de Barcelona 2019 Final Degree Projects (TFG) - Chemistry 1 29 urn:nbn:de:kobv:b43-511626 http://creativecommons.org/licenses/by-nc-nd/3.0/de/deed.de 2020-09-09 OPUS4-47341 misc Sauer, Andreas; Buttler, Sabine; Becker, Roland Bericht zum 24. BAM-Ringversuch "Altlasten" Präsentation der Ergebnisse eines Ringversuchs zur Kompetenzbewertung von Prüflaboratorien auf dem Gebiet der anorganischen und organischen Bodenanalytik: Polychlorierte Biphenyle (PCB) in Boden, Mineralölkohlenwasserstoffe (MKW) in Boden, Elemente in Boden und Gesamtcyanid in Boden. Berlin Bundesanstalt für Materialforschung und -prüfung (BAM) 2018 1 183 urn:nbn:de:kobv:b43-473418 https://creativecommons.org/licenses/by-nc-nd/4.0/deed.de 2019-02-11 OPUS4-58890 misc Gornushkin, Igor; Kornev, R.A.; Shabarova, L.V.; Ermakov, A.A.; Mochalov, G.M.; Rekunov, N.V.; Medov, V.A.; Chaschina, A.D.; Kalinina, A.A. Features of Plasma-Chemical Hydrogen Reduction of Volatile Silicon and Germanium Chlorides in RF-arc Discharge The processes of hydrogen reduction of silicon and germanium chlorides under the conditions of radio-frequency (40.68 MHz) counteracted arc discharge stabilized between two rod electrodes were investigated. The main gas-phase and solid products of plasma-chemical transformations were determined. Thermodynamic analysis of SiCl4 + H2 and GeCl4 + H2 systems was carried out. It is shown that under the implemented experimental conditions, equilibrium components of the products are established. The detected spectra of chemical activity were studied, which gave reason to assume that the molecular mechanism of the hydrogen reduction process is the main one. The impurity composition of gas-phase and solid reaction products was investigated. The possibility of single-stage production of high-purity Si and Ge mainly in the form of compact ingots, as well as high-purity chlorosilanes and trichlorogermane, was shown. 2023 Preprints.org 1 14 urn:nbn:de:kobv:b43-588909 10.20944/preprints202311.1412.v1 https://creativecommons.org/licenses/by/4.0/deed.de 2023-11-27