To tag or not to tag: A comparative evaluation of immunoaffinity-labeling and tandem mass spectrometry for the identification and localization of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation

Posttranslational carbonylation of proteins by the covalent attachment of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is a biomarker of oxidative stress. Tandem mass spectrometry (MS/MS) has become an essential tool for characterization of this modification. Chemical tagging methods have been used to facilitate the immunoaffinity-based enrichment or even quantification of HNE-modified peptides and proteins. With MS/MS spectra of the untagged modified peptides considered as references, a comparative evaluation is presented focusing on the impact of affinity-tagging with four carbonyl-specific reagents (2,4-dinitrophenyl hydrazine, biotin hydrazide, biotinamidohexanoic acid hydrazide and N′-aminooxymethylcarbonyl-hydrazino D-biotin) on collision-induced dissociation of the tagged HNE-carbonylated peptides. Our study has shown that chemical labeling may not be carried out successfully for all the peptides and with all the reagents. The attachment of a tag usually cannot circumvent the occurrence of strong neutral losses observed with untagged species and, in addition, fragmentation of the introduced tag may also happen. Chemical tagging of certain peptides may, nevertheless, afford more sequence ions upon MS/MS than the untagged carbonylated peptide, especially when Michael addition of the lipid peroxidation product occurs on cysteine residues. Therefore, tagging may increase the confidence of identifications of HNE-modified peptides by database searches.

Graphical AbstractTandem mass spectra of untagged peptides covalently modified by 4-hydroxy-2-nonenal and corresponding affinity-tagged species were compared with attention to peptide identification through protein database search.Figure optionsDownload full-size imageDownload high-quality image (45 K)Download as PowerPoint slideHighlights► Tandem mass spectrometry of HNE-modified peptides: untagged and with carbonyl-specific affinity-tags. ► Affinity-tagging may decrease neutral losses of HNE from peptide chain under collision-induced dissociation. ► Increased confidence of identifications from protein database for HNE-modified peptides after affinity-tagging. ► Higher-order tandem mass spectrometry (MS3) or electron-based dissociation also increases confidence of identification.