Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage
✍ Scribed by May Y. Kwong; Reed J. Harris
- Publisher
- Cold Spring Harbor Laboratory Press
- Year
- 2008
- Tongue
- English
- Weight
- 267 KB
- Volume
- 3
- Category
- Article
- ISSN
- 0961-8368
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✦ Synopsis
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.