Protein Science, Vol 3, Issue 1 147-149, Copyright © 1994 by Cold Spring Harbor Laboratory Press

FOR THE RECORD

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

M. Y. KWONG and R. J. HARRIS
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080

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 iso-aspartate 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{deg}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.
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