Targeted ¹⁸o-labeling for improved proteomic analysis of carbonylated peptides by mass spectrometry

Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS²) to peptides within a sequence database. Although effective to some degree, reliable proteomic characterization of carbonylated peptides using this approach remains a challenge needing new, complementary solutions. To this end, we developed a method based on partial ¹⁸O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra of carbonylated peptides and enables their detection without reliance on matching MS² spectra to a peptide sequence. Key to our method were optimized measures for eliminating trypsin-catalyzed incorporation of ¹⁸O at peptide C-termini, and for stabilizing the incorporated ¹⁸O within the carbonyl modification to prevent its loss during liquid chromatography separation. Applying our method to a rat skeletal muscle homogenate treated with the carbonyl modification 4-hyroxynonenal (4-HNE), we demonstrated its compatibility with solid-phase hydrazide enrichment of carbonylated peptides from complex mixtures. Additionally, we demonstrated the value of ¹⁸O isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS² and/or sequence database searching. Combining our ¹⁸O-labeling method with a customized automated software script, we systematically evaluated for the first time the efficiency of MS² and sequence database searching for identifying HNE-modified peptides. We estimated that less than half of the modified peptides selected for MS² were successfully identified. Collectively, our method and software should provide valuable new tools for investigators studying protein carbonylation via mass spectrometry-based proteomics.