Reactive electrophiles formed from toxic drugs and chemicals and by endogenous oxidative stress covalently modify proteins. Although protein covalent binding is thought to initiate a variety of adaptive and toxic responses, the identities of the protein targets are generally unknown, as are protein structural features that confer susceptibility to modification. We have analyzed the protein targets in nuclear and cytoplasmic proteomes from HEK293 cells treated in vitro with two biotin-tagged, thiol-reactive electrophiles, (+)-biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine (PEO-IAB) and 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC). Biotinylated peptides were captured by affinity enrichment using neutravidin beads, and the adducted peptides were then analyzed by multidimensional liquid chromatography-tandem mass spectrometry. A total of 897 adducts were mapped to different cysteine residues in 539 proteins. Adduction was selective and reproducible, and > 90% of all adducted proteins were modified at only one or two sites. A core group of 125 cysteines (14% of the total) was consistently modified by both electrophiles. Selective modification of several protein domain structures and motifs indicates that certain protein families are particularly susceptible to alkylation. This approach can be extended to studies of other protein-damaging oxidants and electrophiles and can provide new insights into targets and consequences of protein damage in toxicity and disease.