Identification of succinimide sites in proteins by N‐terminal sequence analysis after alkaline hydroxylamine cleavage

Open Access
Publisher Website
Google Scholar
Abstract
Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 °C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically ∼ 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site.