title: 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.
abstract: 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.
authors.affiliation: Department of Molecular Biology & Immunology, University of North Texas Health Science Center, Fort Worth, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Guo
authors.forename: Laszlo
authors.initials: L
authors.lastname: Prokai
mesh_terms: D000447:Aldehydes / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000502:physiology; D002478:Cells, Cultured; D006801:Humans; D007118:Immunoassay / Q000379:methods; D015227:Lipid Peroxidation / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D010449:Peptide Mapping / Q000379:methods*; D050050:Protein Carbonylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013194:Staining and Labeling / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D000447:Aldehydes; C027576:4-hydroxy-2-nonenal