Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage. |
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| Authors: | M. Y. Kwong and R. J. Harris |
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| Affiliation: | Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080. |
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| 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 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 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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| Keywords: | deamidation hydroxylamine isoaspartate sequencing succinimide |
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