The effect of histidine oxidation on the dissociation patterns of peptide ions

Oxidative modifications to amino acid side chains can change the dissociation pathways of peptide ions, although these variations are most commonly observed when cysteine and methionine residues are oxidized. In this work we describe the very noticeable effect that oxidation of histidine residues can have on the dissociation patterns of peptide ions containing this residue. A common product ion spectral feature of doubly charged tryptic peptides is enhanced cleavage at the C-terminal side of histidine residues. This preferential cleavage arises as a result of the unique acid/base character of the imidazole side chain that initiates cleavage of a proximal peptide bond for ions in which the number of protons does not exceed the number of basic residues. We demonstrate here that this enhanced cleavage is eliminated when histidine is oxidized to 2-oxo-histidine because the proton affinity and nucleophilicity of the imidazole side chain are lowered. Furthermore, we find that oxidation of histidine to 2-oxo-histidine can cause the misassignment of oxidized residues when more than one oxidized isomer is simultaneously subjected to tandem mass spectrometry (MS/MS). These spectral misinterpretations can usually be avoided by using multiple stages of MS/MS (MS(n)) or by specially optimized liquid chromatographic separation conditions. When these approaches are not accessible or do not work, N-terminal derivatization with sulfobenzoic acid avoids the problem of mistakenly assigning oxidized residues.