Mass spectrometry-based characterization of posttranslational modifications by 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (HNE), an alpha,beta-unsaturated aldehyde, generated by the peroxidation of polyunsaturated membrane lipids can covalently modify cellular target proteins thereby affecting their biological structure and function. Elucidation of the chemistry of the reactions of HNE with proteins is fundamental to understanding the mechanism of HNE-induced cytotoxicity.;The main focus of this study is to develop mass spectrometric-based methods for the unambiguous identification of HNE-carbonylated proteins, including precise determination of the position of their adductions. In this study, we have demonstrated that high mass resolving power and mass measurement accuracy of the Fourier transform ion cyclotron resonance mass spectrometer facilitated the detailed compositional analysis of the covalent adducts of HNE to apomyoglobin without further need for the deconvolution and transformation. We have also shown the value of collision induced dissociation (CID) and electron capture dissociation (ECD) methods of peptide fragmentation to characterize protein modification by HNE. The characteristic elimination of HNE (156 Da) during CID-based fragmentation of HNE-modified peptides serves as a signature tag for the HNE modified peptides. The ability to retain the labile HNE moiety of the peptides during ECD-based method of dissociation assists in localization of the exact sites of HNE carbonylation.;We have also introduced data-dependent neutral loss (NL)-driven MS3 and NL-triggered ECD tandem mass spectrometry methods for the characterization of HNE-modification sites in peptides. The NL-driven MS3 method enabled identification of, in addition to Michael adducts, the Schiff-base adducts in proteins which represent <1% of HNE protein modifications. We have observed that the selective enrichment of HNE-modified peptides prior to mass spectrometric analysis has the added advantage of producing enhanced data quality of MS3 spectrum by increasing the number of ion counts for fragmentation. Furthermore, the enrichment technique also reduces the acquisition of false MS3 spectra triggered due to isobaric fragment ions produced during CID. Our developed methods will be useful for our future in vivo studies that aim for the characterization of HNE and other carbonyl modifications in proteins extracted from various complex matrices such as cells and tissues and this may potentially lead to the identification of new biomarkers of carbonyl stress.