TY - JOUR A1 - Tscheuschner, Georg A1 - Kaiser, Melanie N. A1 - Lisec, Jan A1 - Beslic, Denis A1 - Muth, Thilo A1 - Krüger, Maren A1 - Mages, Hans Werner A1 - Dorner, Brigitte G. A1 - Knospe, Julia A1 - Schenk, Jörg A. A1 - Sellrie, Frank A1 - Weller, Michael G. T1 - MALDI-TOF-MS-based identification of monoclonal murine Anti-SARS-CoV-2 antibodies within one hour JF - Antibodies N2 - 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 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to 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 60 min 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 degrees 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. 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. KW - SARS-CoV-2 antibody KW - reproducibility crisis KW - peptide mass KW - fingerprinting KW - monoclonal antibody KW - traceability KW - identity KW - antibody KW - identification KW - antibody light chain KW - MALDI-TOF-MS Y1 - 2022 U6 - https://doi.org/10.3390/antib11020027 SN - 2073-4468 VL - 11 IS - 2 PB - MDPI CY - Basel ER - TY - JOUR A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Waldbach Braga, Tess A1 - Rackiewicz, Michal A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.) JF - Processes : open access journal N2 - Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples. KW - wheat KW - α-amylase/trypsin inhibitors KW - fractionation KW - purification KW - reversed-phase chromatography KW - ion-exchange chromatography KW - design of experiment KW - LC–MS/MS KW - MALDI-TOF-MS Y1 - 2022 U6 - https://doi.org/10.3390/pr10020259 SN - 2227-9717 VL - 10 SP - 1 EP - 18 PB - MDPI CY - Basel, Schweiz ET - 2 ER - TY - JOUR A1 - Khozroughi, Amin Ghadiri A1 - Braga, Tess Waldbach A1 - Wagner, Janine A1 - Rawel, Harshadrai Manilal T1 - Investigation of the post mortem zinc protoporphyrin IX fluorescence with respect to its protein-bound and unbound occurrence in aqueous meat extracts JF - Food chemistry N2 - Zinc protoporphyrin IX (ZnPP) is known to accumulate in most meat products during storage. However, the pathway of its formation is not yet completely clarified. To gain more insights into the specificity of ZnPP occurrence, a SEC-HPLC-UV-fluorescence setup was established to screen the proteins in aqueous meat extracts for their ZnPP fluorescence during incubation. In accordance with previous studies it was identified by SDS-PAGE and MALDI-TOF-MS that ZnPP formation takes place in myoglobin. In this study, valuable new insights into the ZnPP forming pathway were gained, as our results indicated that a significant part of ZnPP - after being formed within the protein - is transitioned into free ZnPP during incubation. Additionally, the obtained results implied that ZnPP may also occur in proteins of higher molecular weight (> 100 kDa). KW - Meat KW - Fluorescence screening KW - Post mortem chemistry KW - SDS-PAGE KW - SEC-HPLC KW - MALDI-TOF-MS Y1 - 2019 U6 - https://doi.org/10.1016/j.foodchem.2019.01.080 SN - 0308-8146 SN - 1873-7072 VL - 283 SP - 462 EP - 467 PB - Elsevier CY - Oxford ER -