Journal of the American Society for Mass Spectrometry

, Volume 19, Issue 6, pp 855–864

Use of 18O labels to monitor deamidation during protein and peptide sample processing

  • Xiaojuan Li
  • Jason J. Cournoyer
  • Cheng Lin
  • Peter B. O’Connor
Articles

DOI: 10.1016/j.jasms.2008.02.011

Cite this article as:
Li, X., Cournoyer, J.J., Lin, C. et al. J Am Soc Mass Spectrom (2008) 19: 855. doi:10.1016/j.jasms.2008.02.011

Abstract

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c · + 58 and z − 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)(n−1)+· − 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of 18O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (∼4 h) trypsin digestion process generates no additional detectable peptide deamidations.

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Copyright information

© American Society for Mass Spectrometry 2008

Authors and Affiliations

  • Xiaojuan Li
    • 1
  • Jason J. Cournoyer
    • 2
  • Cheng Lin
    • 1
  • Peter B. O’Connor
    • 1
  1. 1.Mass Spectrometry Resource, Department of BiochemistryBoston University School of MedicineBostonUSA
  2. 2.Department of ChemistryBoston UniversityBostonUSA