Mass spectrometry with or without pre-analysis peptide fractionation can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen and electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighboring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4-hydroxynonenal (4HNE) of human bile acid CoA: amino acid N-acyltransferase (hBAT) and the cytosolic brain isoform of creatine kinase (CKBB) is associated with site-specific modifications. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) using nanoLC-electrospray ionization-mass spectrometry (ESI-MS) or direct infusion-ESI-MS with gas phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB, respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.