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

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. Copyright © 1994 The Protein Society