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Peptide pools consist of short amino acid sequences and have proven to be versatile tools in various research areas in immunology and clinical applications. They are commercially available in many different compositions and variants. However, unlike other reagents that consist of only one or a few compounds, peptide pools are highly complex products which makes their quality control a major challenge. Quantitative peptide analysis usually requires sophisticated methods, in most cases isotope-labeled standards and reference materials. Usually, this would be prohibitively laborious and expensive. Therefore, an approach is needed to provide a practical and feasible method for quality control of peptide pools. With insufficient quality control, the use of such products could lead to incorrect experimental results, worsening the well-known reproducibility crisis in the biomedical sciences. Here we propose the use of ultra-high performance liquid chromatography (UHPLC) with two detectors, a standard UV detector at 214 nm for quantitative analysis and a high-resolution mass spectrometer (HRMS) for identity confirmation. To be cost-efficient and fast, quantification and identification are performed in one chromatographic run. An optimized protocol is shown, and different peak integration methods are compared and discussed. This work was performed using a peptide pool known as CEF advanced, which consists of 32 peptides derived from cytomegalovirus (CMV), Epstein–Barr virus (EBV) and influenza virus, ranging from 8 to 12 amino acids in length.
DNA long-term stability and integrity is of importance for applications in DNA based bio-dosimetry, data-storage, pharmaceutical quality-control, donor insemination and DNA based functional nanomaterials. Standard protocols for these applications involve repeated freeze-thaw cycles of the DNA, which can cause detrimental damage to the nucleobases, as well as the sugar-phosphate backbone and therefore the whole molecule. Throughout the literature three hypotheses can be found about the underlying mechanisms occurring during freeze-thaw cycles. It is hypothesized that DNA single-strand breaks during freezing can be induced by mechanical stress leading to shearing of the DNA molecule, by acidic pH causing damage through depurination and beta elimination or by the presence of metal ions catalyzing oxidative damage via reactive oxygen species (ROS). Here we test these hypotheses under well defined conditions with plasmid DNA pUC19 in high-purity buffer (1xPBS) at physiological salt and pH 7.4 conditions, under pH 6 and in the presence of metal ions in combination with the radical scavengers DMSO and Ectoine. The results show for the 2686 bp long plasmid DNA, that neither mechanical stress, nor pH 6 lead to degradation during repeated freeze-thaw cycles. In contrast, the presence of metal ions (Fe2+) leads to degradation of DNA via the production of radical species.
MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour
(2022)
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.
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.
More than 80 representatives of SMEs, industrial companies and research institutes met on September 12 at the workshop "Challenges in Additive Manufacturing: Innovative Materials and Quality Control" at BAM in Adlershof to discuss the latest developments in materials and quality control in additive manufacturing.
In special lectures, researchers, users and equipment manufacturers reported on the latest and future developments in additive manufacturing. Furthermore, funding opportunities for projects between SMEs and research institutions on a national and European level were presented.
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.
Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the sticky character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing an LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend an LC-MS/MS-based quantification method with a dynamic range of 0.5–40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials.