Arthur Hinterholzer, Vesna Stanojlovic, Christof Regl, Christian G. Huber, Chiara Cabrele, Mario Schubert
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引用次数: 10
Abstract
The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of?+?18?Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D?NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pKa values of isoAsp and C-terminal Asp in short peptides. The characteristic 1H-13C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4–5) and 40?°C. The results show that the application of our 2D?NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.
期刊介绍:
The Journal of Biomolecular NMR provides a forum for publishing research on technical developments and innovative applications of nuclear magnetic resonance spectroscopy for the study of structure and dynamic properties of biopolymers in solution, liquid crystals, solids and mixed environments, e.g., attached to membranes. This may include:
Three-dimensional structure determination of biological macromolecules (polypeptides/proteins, DNA, RNA, oligosaccharides) by NMR.
New NMR techniques for studies of biological macromolecules.
Novel approaches to computer-aided automated analysis of multidimensional NMR spectra.
Computational methods for the structural interpretation of NMR data, including structure refinement.
Comparisons of structures determined by NMR with those obtained by other methods, e.g. by diffraction techniques with protein single crystals.
New techniques of sample preparation for NMR experiments (biosynthetic and chemical methods for isotope labeling, preparation of nutrients for biosynthetic isotope labeling, etc.). An NMR characterization of the products must be included.