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. 1994 Jan;3(1):147–149. doi: 10.1002/pro.5560030119

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

M Y Kwong 1, R J Harris 1
PMCID: PMC2142483  PMID: 8142891

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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Selected References

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  1. Bischoff R., Lepage P., Jaquinod M., Cauet G., Acker-Klein M., Clesse D., Laporte M., Bayol A., Van Dorsselaer A., Roitsch C. Sequence-specific deamidation: isolation and biochemical characterization of succinimide intermediates of recombinant hirudin. Biochemistry. 1993 Jan 19;32(2):725–734. doi: 10.1021/bi00053a042. [DOI] [PubMed] [Google Scholar]
  2. Bornstein P., Balian G. Cleavage at Asn-Gly bonds with hydroxylamine. Methods Enzymol. 1977;47:132–145. doi: 10.1016/0076-6879(77)47016-2. [DOI] [PubMed] [Google Scholar]
  3. Carter P., Presta L., Gorman C. M., Ridgway J. B., Henner D., Wong W. L., Rowland A. M., Kotts C., Carver M. E., Shepard H. M. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4285–4289. doi: 10.1073/pnas.89.10.4285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Di Donato A., Ciardiello M. A., de Nigris M., Piccoli R., Mazzarella L., D'Alessio G. Selective deamidation of ribonuclease A. Isolation and characterization of the resulting isoaspartyl and aspartyl derivatives. J Biol Chem. 1993 Mar 5;268(7):4745–4751. [PubMed] [Google Scholar]
  5. Geiger T., Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem. 1987 Jan 15;262(2):785–794. [PubMed] [Google Scholar]
  6. George-Nascimento C., Lowenson J., Borissenko M., Calderón M., Medina-Selby A., Kuo J., Clarke S., Randolph A. Replacement of a labile aspartyl residue increases the stability of human epidermal growth factor. Biochemistry. 1990 Oct 16;29(41):9584–9591. doi: 10.1021/bi00493a012. [DOI] [PubMed] [Google Scholar]
  7. Johnson B. A., Shirokawa J. M., Hancock W. S., Spellman M. W., Basa L. J., Aswad D. W. Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone. J Biol Chem. 1989 Aug 25;264(24):14262–14271. [PubMed] [Google Scholar]
  8. Kossiakoff A. A. Tertiary structure is a principal determinant to protein deamidation. Science. 1988 Apr 8;240(4849):191–194. doi: 10.1126/science.3353715. [DOI] [PubMed] [Google Scholar]
  9. Morimoto K., Inouye K. Single-step purification of F(ab')2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW. J Biochem Biophys Methods. 1992 Mar;24(1-2):107–117. doi: 10.1016/0165-022x(92)90051-b. [DOI] [PubMed] [Google Scholar]
  10. Potter S. M., Henzel W. J., Aswad D. W. In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV. Protein Sci. 1993 Oct;2(10):1648–1663. doi: 10.1002/pro.5560021011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Stephenson R. C., Clarke S. Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. J Biol Chem. 1989 Apr 15;264(11):6164–6170. [PubMed] [Google Scholar]
  12. Tyler-Cross R., Schirch V. Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. J Biol Chem. 1991 Nov 25;266(33):22549–22556. [PubMed] [Google Scholar]
  13. Violand B. N., Schlittler M. R., Kolodziej E. W., Toren P. C., Cabonce M. A., Siegel N. R., Duffin K. L., Zobel J. F., Smith C. E., Tou J. S. Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129. Protein Sci. 1992 Dec;1(12):1634–1641. doi: 10.1002/pro.5560011211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Violand B. N., Schlittler M. R., Toren P. C., Siegel N. R. Formation of isoaspartate 99 in bovine and porcine somatotropins. J Protein Chem. 1990 Feb;9(1):109–117. doi: 10.1007/BF01024992. [DOI] [PubMed] [Google Scholar]
  15. Voorter C. E., de Haard-Hoekman W. A., van den Oetelaar P. J., Bloemendal H., de Jong W. W. Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate. J Biol Chem. 1988 Dec 15;263(35):19020–19023. [PubMed] [Google Scholar]
  16. Wright H. T. Sequence and structure determinants of the nonenzymatic deamidation of asparagine and glutamine residues in proteins. Protein Eng. 1991 Feb;4(3):283–294. doi: 10.1093/protein/4.3.283. [DOI] [PubMed] [Google Scholar]

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