Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming

Formation of atypical isoaspartyl (isoAsp) sites in peptides and proteins via the deamidation-linked isomerization of asparaginyl–Xaa bonds or direct isomerization of aspartyl–Xaa bonds is a major contributor to spontaneous protein damage under mild conditions. This nonenzymatic reaction reroutes the Asx–Xaa peptide bond through the β-carbonyl of asparaginyl or aspartyl residues, thereby adding an extra carbon to the polypeptide backbone. Formation of isoAsp has been implicated in protein inactivation, aggregation, degradation, and autoimmunity. Knowing the location of isoAsp sites in proteins is important for understanding mechanisms of protein damage and for characterizing protein pharmaceuticals. Here we present a simple nonradioactive method for direct localization of isoAsp residues in peptides or proteins. Using three model peptides, we demonstrate that isoAsp linkages can be cleaved selectively and in high yield by a two-step process in which (i) the isoAsp linkage is converted into a succinimide on incubation with S-adenosyl-l-methionine and the commercially available enzyme, protein l-isoaspartyl-O-methyltransferase, and (ii) the succinimidyl bond is then cleaved by hydroxylamine under conditions that minimize cleavage of the traditional hydroxylamine-sensitive Asn–Gly and related peptide bonds. Location of the isoAsp linkage is then inferred by identifying the cleavage products by mass spectrometry or N-terminal sequencing.