Analysis of d-β-Aspartyl Isomers at Specific Sites in Proteins

  • Noriko FujiiEmail author
  • Norihiko Fujii
Part of the Methods in Molecular Biology book series (MIMB, volume 794)


Recent studies have shown that biologically uncommon d-β-aspartic acid residues accumulate in specific proteins during the aging process. However, aspartyl residues are not racemized uniformly because d-Asp appears to be confined to particular sites in these proteins. We here describe the method to identify the specific sites of d-β-aspartic acids inversion in proteins.

Key words

d-Amino acid β-Aspartic acid Aging RP-HPLC Mass spectrometry Enantiomer Isomer Lens crystallin 



This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


  1. 1.
    Helfman P M, Bada J L (1975) Aspartic acid racemization in tooth enamel from living humans. Proc Natl Acad Sci USA 72, 2891–2894.PubMedCrossRefGoogle Scholar
  2. 2.
    Ohtani S et al. (1998) Changes in the amount of D-aspartic acid in the human femur with age. Growth Dev Aging 62, 141–148.PubMedGoogle Scholar
  3. 3.
    Cloos P A, Fledelius C (2000) Collagen fragments in urine derived from bone resorption are highly racemized and isomerized: a biological clock of protein aging with clinical potential. Biochem J 345, 473–480.PubMedCrossRefGoogle Scholar
  4. 4.
    Powell J T, Vine N, Crossman M (1992) On the accumulation of D-aspartate in elastin and other proteins of the ageing aorta. Atherosclerosis 97, 201–208.PubMedCrossRefGoogle Scholar
  5. 5.
    Ritz-Timme S, Laumeier I, Collins M (2003) Age estimation based on aspartic acid racemization in elastin from the yellow ligaments. Int J Legal Med 117, 96–101.PubMedGoogle Scholar
  6. 6.
    Fisher G H et al. (1992) Quantification of D-aspartate in normal and Alzheimer brains. Neurosci Lett 143, 215–218.PubMedCrossRefGoogle Scholar
  7. 7.
    Shapira R, Chou C H (1987) Differential racemization of aspartate and serine in human myelin basic protein. Biochem Biophys Res Commun 146, 1342–1349.PubMedCrossRefGoogle Scholar
  8. 8.
    Roher A E et al. (1993) Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer’s disease. J Biol Chem 268, 3072–3083.PubMedGoogle Scholar
  9. 9.
    Masters P M, Bada J L, Zigler J S Jr (1977) Aspartic acid racemisation in the human lens during ageing and in cataract formation. Nature 268, 71–73.PubMedCrossRefGoogle Scholar
  10. 10.
    Fujii N et al. (1994) Simultaneous stereoinversion and isomerization at specific aspartic acid residues in alpha A-crystallin from aged human lens. J. Biochem 116, 663–669.PubMedGoogle Scholar
  11. 11.
    Fujii N et al. (1994) Simultaneous racemization and isomerization at specific aspartic acid residues in alpha B-crystallin from the aged human lens. Biochim Biophys Acta 1204, 157–163.PubMedCrossRefGoogle Scholar
  12. 12.
    Kaji Y et al. (2007) Localization of D-beta-aspartic acid-containing proteins in human eyes. Invest Ophthalmol Vis Sci 48, 3923–3927.PubMedCrossRefGoogle Scholar
  13. 13.
    Kaji Y et al. (2009) Immunohistochemical localisation of D-beta-aspartic acid in pingueculae. Br J Ophthalmol 93, 974–976.PubMedCrossRefGoogle Scholar
  14. 14.
    Kaji Y et al. (2009) Immunohistochemical localisation of D-beta-aspartic acid-containing proteins in climatic droplet keratopathy. Br J Ophthalmol 93, 977–979.PubMedCrossRefGoogle Scholar
  15. 15.
    Fujii N et al. (2002) The presence of D-beta-aspartic acid-containing peptides in elastic fibers of sun-damaged skin: a potent marker for ultraviolet-induced skin aging. Biochem Biophys Res Commun 294, 1047–1051.PubMedCrossRefGoogle Scholar
  16. 16.
    Young G W et al. (2005) Protein L-isoaspartyl methyltransferase catalyzes in vivo racemization of Aspartate-25 in mammalian histone H2B. J Biol Chem 280, 26094–26098.PubMedCrossRefGoogle Scholar
  17. 17.
    Laemmli U K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  18. 18.
    Yamazaki Y et al. (2010) Differentiation and semiquantitative analysis of an isoaspartic acid in human alpha-Crystallin by postsource decay in a curved field reflectron. Anal Chem 82, 6384–6394.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Research Reactor InstituteKyoto UniversityOsakaJapan

Personalised recommendations