Protein damage by reactive electrophiles: targets and consequences

Abstract

It has been 60 years since the Millers first described the covalent binding of carcinogens to tissue proteins. Protein covalent binding was gradually overshadowed by the emergence of DNA adduct formation as the dominant paradigm in chemical carcinogenesis but re-emerged in the early 1970s as a critical mechanism of drug and chemical toxicity. Technology limitations hampered the characterization of protein adducts until the emergence of mass spectrometry-based proteomics in the late 1990s. The time since then has seen rapid progress in the characterization of the protein targets of electrophiles and the consequences of protein damage. Recent integration of novel affinity chemistries for electrophile probes, shotgun proteomics methods, and systems modeling tools has led to the identification of hundreds of protein targets of electrophiles in mammalian systems. The technology now exists to map the targets of damage to critical components of signaling pathways and metabolic networks and to understand mechanisms of damage at a systems level. The implementation of sensitive, specific analyses for protein adducts from both xenobiotic-derived and endogenous electrophiles offers a means to link protein damage to clinically relevant health effects of both chemical exposures and disease processes.

Figure 1

Visible spectra of the carcinogens 3-methyl-4-dimethylaminoazobenzene, its N- monomethyl derivative and the protein-bound chromophores formed in liver following administration of the same compounds to rats in vivo. From reference (2).

Figure 2

Detection and identification of protein adducts using SDS-PAGE and western blotting with antibodies against adducts (top). Application of 2D-SDS-PAGE to resolution and detection of protein adducts (bottom). The example depicts use or radiolabel to locate adducts, although western blotting with antibodies against adducts is also used (see text for discussion).

Figure 3

Biotin electrophile probes used for shotgun LC-MS-MS analysis of cellular protein targets.

Figure 4

Strategies for analyses of proteins and peptide sequence targets modified by biotinylated electrophile probes. Capture of intact protein adducts (A) followed by digestion and LC-MS-MS analysis primarily yields unmodified peptides, which enables identification of protein targets, but results in few adduct identifications. Capture of adducted peptides following proteolytic digestion (B) results in an enriched adduct population for LC-MS-MS and identifies greater numbers of specific adduct sites.

Figure 5

Click chemistry for post-reaction tagging of alkynyl-labeled adducts with biotin.