Identification of Asp Isomerization in Proteins by 18O Labeling and Tandem Mass Spectrometry

Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H ₂ ¹⁸ O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹⁶ O-containing counterparts, due to ¹⁸ O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹⁸ O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.