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Chemical modification of proteins and polynucleotides by electrophilic products of lipid oxidation, generated under condition of oxidative stress, have been implicated in a variety of diseases, such as atherosclerosis, neurodegenerative disease, liver disease, and cancer, to name a few. Most work has focused on reactive bifunctional aldehydes like malondialdehyde, 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and 4,5-epoxy-2-alkenals, in each case with an eye on modification and cross-linking of protein-based lysine, histidine, and cysteine residues.The reactions of HNE and ONE with two model proteins, RNase and β-lactoglobulin, and their Lys ε-dimethylamino derivatives, studied through spectrophotometry, showed that ONE was 6-31 times more reactive than HNE with these proteins. The fastest reaction of ONE with proteins involves Schiff base formation at Lys ε-amino groups, whereas Schiff base formation is not spectroscopically apparent for HNE. Detailed kinetic studies of the initial reaction of HNE and ONE were carried out with amino acids and amino acid surrogates. Whereas the reactions with imidazole and thiol nucleophiles involve straightforward Michael adduct formation, kinetics analyses revealed the reversibility of both HNE Michael addition to amine and ONE Schiff base formation with amine. Although ONE is more reactive than HNE toward conjugate addition of imidazole and thiol nucleophiles, it is less reactive than HNE toward Lys/amine Michael adduction. Moreover, ONE is 4-5 times more cytotoxic than HNE to human neuroblastoma cells at concentrations near the threshold of lethality. The greater neurotoxicity of ONE could reflect in part the different reactivity characteristics of ONE as compared to HNE.Mass spectrometric studies (MALDI-TOF, HPLC-MS/MS) revealed that apomyoglobin was modified predominantly by isomeric epoxyketooctadecenoic acid (EKODE) residues when the protein was exposed to mild non-enzymatic oxidation of linoleic acid. Six regio- and stereoismoers of EKODE were independently synthesized and identified by HPLC in the system of autoxidation of linoleic acid catalyzed by Fe(II)/ascorbic acid. The nature of EKODE-His adduct formation was defined through model studies to reflect Michael addition of the His imidazole group. In addition, kinetic studies revealed that the various EKODE isomeric families show significantly different reactivity toward imidazole nucleophiles.4,5-Epoxy-2-alkenals, another family of lipoxidation end products, can modify proteins and give rise to Lys-Lys protein cross-linking in the form of lipofuscin-like polypyrroles. Here a novel Lys-His cross-linking piperidine structure was identified in the reaction of trans-4,5-epoxy-2(E)-heptenal (EH) with simple amine and imidazole surrogates for the Lys and His side-chains of proteins. Protein cross-linking studies indicated that Lys-His cross-linking should play a more important role than Lys-Lys cross-linking reported before.Neurofilament proteins (NFs) are among the major cytoskeletal proteins in axons that are found to be the major targets of oxidative modification. There are many Lys-Ser-Pro (KSP) repeats in the C-termini of neurofilament heavy subunit (NFH) and neurofilament medium subunit (NFM). Recent studies revealed that neurofilament lysine modification by HNE appears to be regulated by the phosphorylation state of the adjacent serine. To investigate the possibility of an intrinsic chemical difference caused by the presence or absence of Ser phosphate adjacent to Lys in a simple model system, small KS-containing peptides in both phospho forms were prepared and studied with HNE and its analog 4-oxononanal. The results indicated that phosphorylation of the Ser residue does not increase the adjacent lysine reactivity to HNE in small peptides, and the high reactivity of phosphorylated NFH may be due to alteration of the protein conformation that induces high Lys reactivity.Previous studies on the modification of proteins by products of lipid-peroxidation have led to the recognition of a number of biomarkers that can be detected in various tissues in a variety of disease states associated with oxidative stress. The work in this thesis has revealed additional information on the nature of such protein modification, and may lead to new biomarkers with distinct features. These efforts should ultimately help define the role of lipoxidation-derived protein modifications in disease pathogenesis, in particular whether such modifications play at least in part a causative role or a merely as a disease-associated biomarker.