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Default Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy

Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy

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 ofâ??+â??18Â*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 2DÂ*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 4â??5) and 40Â*°C. The results show that the application of our 2DÂ*NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.



Source: Journal of Biomolecular NMR
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