Investigating peroxide-induced Asp epimerization in peroxiredoxin 2 using enzymatic hydrolysis and LC-MS/MS.

Stereoinversion of amino acids from the -form to the -form alters protein structure and, possibly, biological activity. This modification causes no change in protein m/z and is often overlooked during protein analysis. Aspartic acid epimerization (AAE) occurs faster than other amino acids and is known to be accelerated by free radicals and peroxides. Herein, we propose a two-step enzymatic hydrolysis using chymotrypsin followed by aminopeptidase M, combined with LC-MS/MS analysis, for the selective detection of -Asp in Peroxiredoxin 2 (Prx2), a key antioxidant enzyme, after reacting with tert-butyl hydroperoxide (TBHP). Additionally, we have developed an LC-MS/MS method for the separation and quantification of YPLDF and YPLDF peptides located in the Prx2 active site. Our results demonstrate that treatment of isolated Prx2 with TBHP results in epimerization at Asp, with the epimerization increasing over the reaction period. A similar time-dependent epimerization of Asp was observed when whole RBCs were exposed to TBHP, followed by the isolation of Prx2. Notably, Asp is located in the Prx2 active site, and the chirality change of Asp to the -form at this position leads to a decrease in binding affinity with the target protein. Further studies are needed to definitively link oxidation-driven alterations in the protein stereochemistry of Prx2 to functional changes and oxidative stress.