Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

Identification of isoaspartylated peptides by electron-transfer/higher-energy collision dissociation (#86)

Clifford Young 1 2 , Frank Kjeldsen 3 , Michael L Nielsen 1
  1. The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
  2. Future Industries Institute, University of South Australia, Adelaide, South Australia, Australia
  3. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

Isoaspartic acid is a post-translational modification that occurs as a result of deamidation or isomerisation reactions. Although numerous MS methodologies can characterise these subtle protein modifications, isoaspartylation sites are commonly identified by electron-transfer dissociation (ETD) due to the production of c+57 and z-57 diagnostic ions that pinpoint the site of modification. However, ETD is largely inefficient for doubly protonated peptides, which is unfortunately the predominant charge state of protonated tryptic peptides after electrospray ionization. We performed electron-transfer dissociation with supplemental collisional activation or electron-transfer/higher-energy collision dissociation (EThcD) on four doubly charged monoisoaspartylated peptides to see if the overall sequence coverage and/or isoaspartic acid discrimination was improved when compared to ETD alone. Although all three methods produced z-57 ions that confidently located the isoaspartic acid, only EThcD produced MS/MS spectra rich in additional y ions that allowed the peptide sequences to be extensively characterised. The use of EThcD permits the identification of isoaspartylated peptides when their physicochemical properties may be unsuitable for ETD.