4-Oxo-2-nonenal (ONE) is a highly reactive aldehyde derived from endogenous lipid peroxidation, which is associated with the development and progression of chronic human diseases. Despite the fact that ONE exerts biological effects mainly by modifying nucleophilic amino acid residues on proteins in cells, whether and how this bifunctional electrophile forms covalent crosslinks in complex proteomes remains unknown. Here we report a reanalysis of the data previously reported where in situ ONE adduction chemistry has been surveyed by chemoproteomics (Sun et al., Mol Cell Proteomics, 2017), revealing that vicinal Cys and Lys residues on cellular proteins can be directly crosslinked by ONE via initial Michael addition and subsequent Paal-Knorr condensation. Furthermore, we adapted our chemoproteomics platform to enable more efficient and robust crosslinking-mass spectrometry analysis, resulting in the identification of a total of 165 pyrrole-based Cys-Lys crosslinks on 145 proteins from intact cells. Quantitative analysis reveals the dynamic nature of these crosslinks in situ, which is similar to most ONE-derived modifications (i.e., lysine Schiff base adducts, lysine ketoamide adducts, cysteine pyrrole adducts). Biochemical analyses further highlight the functional importance of one of the identified crosslinking events; an inter-molecular crosslink between cofilin and actin proteins might partially be involved in apoptosis induced by this cytotoxic electrophile. Taken together, our study not only expands the inventory of ONE-adducts in cells, but also provides additional mechanistic insights into the potent cytotoxicity by this endogenous electrophile.
Cheng et al. (Tue,) studied this question.