Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.
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.
Kwong
M Y
MY
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.
Harris
R J
RJ
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism*; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D013388:Succinimides / Q000032:analysis* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D006898:Hydroxylamines; D013388:Succinimides; C032620:succinimide; D019811:Hydroxylamine; D014357:Trypsin