UC Riverside

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate formed during reaction of l-isoAsp-containing peptides with PIMT and S-adenosyl methionine (SAM) is reactive with Tris base and results in a Tris-modified aspartic acid residue with a mass shift of +103 Da. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that Tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.