Complete NMR assignment of succinimide, its detection and quantification in peptides and intact proteins
Luigi Grassi, Christof Regl, Sabrina Wildner, Gabriele Gadermaier, Christian G. Huber, Chiara Cabrele, and Mario Schubert
Abstract:
Detecting and quantifying posttranslational modifications (PTMs) in full-length proteins is a challenge, especially in the case of spontaneously occurring, non-enzymatic PTMs. Such a PTM is the formation of succinimide (Snn) in a protein that occurs spontaneously in prone primary sequences and leads typically to an equilibrium between Snn and its hydrolysis products isoaspartate (isoAsp) and aspartate. In order to detect these modifications in proteins by NMR spectroscopy chemical shift assign-ments of reference compounds are required. We used peptide synthesis and 2D NMR spectroscopy to assign all 1H and 13C chemi-cal shifts of Snn and isoAsp and found characteristic chemical shift correlations. To provide chemical shift reference data suitable for comparison with data of denatured proteins, we repeated the assignment in 7 M urea (pH 2.3) and in DMSO. Most characteristic of Snn are the two downfield shifted carbonyl chemical shifts, the chemical shift correlations of Cβ-Hβ of Snn and Cα-Hα of the succeeding residue which are clearly distinct from random coil chemical shift correlations. The characteristic 2D NMR fingerprints of Snn were used to detect and quantify this PTM in the model protein lysozyme, the biotherapeutic filgrastim and the Fc part of immunoglobulin G1. Mass spectrometry (MS) was applied as an additional independent method. The orthogonality of the NMR and MS techniques allows cross-validation, which is especially important to search for subtle PTMs in proteins. Studying PTMs by NMR spectroscopy is a promising method to analyze proteins and peptides either from natural sources, recombinant expression or chemical synthesis.
The open access article can be found  here and  here.

Reviewed by Angela Risch