Chromatographic behavior of peptides containing oxidized methionine residues in proteomic LC–MS experiments: Complex tale of a simple modification

Abstract

On average, the oxidation of a single Met residue to Mso (methionine S-oxide, methionine sulfoxide) and Msn (methionine S,S-dioxide, methionine sulfone) decreases peptide retention in RP HPLC by 2.37 and 1.95 Hydrophobicity Index units (% acetonitrile), respectively. At the same time, the magnitude of the retention shift varies greatly (−9.1 to +0.4% acetonitrile for Mso) depending on peptide sequence. The latter effects are mostly associated with the stabilization of secondary structures upon peptide interaction with the hydrophobic stationary phase: when an oxidized residue is located in the hydrophobic face of an amphipathic helix, the decrease in retention is profound. The same amino acid positioning leads to complete or partial resolution of pairs of peptides containing diastereomeric Mso residues. Contrary to all previously reported observations, and the nature of this modification, we also demonstrate for the first time that methionine oxidation may increase peptide hydrophobicity. This behavior is characteristic for Met residues in the N3 position of an N-capping box stabilization motif prior to the amphipathic helix. All these findings indicate that the prediction of peptide secondary structures upon interaction with hydrophobic surfaces must become an integral part of peptide retention modeling in proteomic applications going forward.