Isoaspartate forms spontaneously from Asn deamidation or Asp isomerization, which can plague protein purification and storage processes. Indeed, it often comes with deleterious consequences, from a loss of function to a gain of toxic properties. IsoAsp detection is not straightforward, notably because it causes weak mass shifts, i.e., +1 or 0 from the native Asn or Asp, respectively. NMR spectroscopy might help in nontargeted detection of isoAsp, but information on isoAsp NMR fingerprint and sensitive detection methods were missing. Here, we report the NMR characterization of isoAsp in ten model, random coil hexapeptides, and release reference chemical shifts and scalar couplings of backbone nuclei from (i-1)-(i)-(i+1) residues (from 283 to 310 K). We show how isoAsp chemical shifts evolve with pH (from 2 to 8) and urea concentration (from 0 to 8 M). This led us to draw methods to identify isoAsp in 13C/15N-enriched and in natural abundance polypeptides. In the latter case, we use notably trypsinization, protein denaturation, and 1H-only NMR, enabling the detection of 10 nmol of isoAsp-containing protein in 1 h. We exemplify this approach on therapeutic products like insulin or the monoclonal antibody trastuzumab.
Julien et al. (2026) studied this question.