We report that protein adducts of isolevuglandin E2 (isoLGE2), a highly reactive product of free radical-induced lipid oxidation, accumulate in human glaucomatous trabecular meshwork (TM) but not in controls. Reactive oxygen species play a pathogenic role in primary open angle glaucoma by fostering changes that reduce permeability of the TM tissue and consequently impede aqueous humor outflow resulting in elevated intraocular pressure. IsoLGs covalently modify proteins and are especially effective in causing protein-protein cross-linking. We found elevated levels of calpain-1 in glaucomatous TM. However, calpain activity in glaucomatous TM is only about 50% of that in controls. This paradox is explicable by the fact that modification by isoLGs renders calpain-1 inactive. Thus, treatment of calpain-1 with isoLGE2 in vitro results in covalent modification, inactivation, the formation of high molecular weight aggregates (as determined by Western and dynamic light scattering analyses), and resistance to proteasomal digestion. IsoLGE2-modified calpain-1 undergoes ubiquitination, and its loading impairs the cellular proteasome activity, consistent with competitive inhibition and formation of suicidal high molecular weight aggregates. These data suggest that interference with proteasomal activity, owing to protein modification by isoLGs, could contribute to glaucoma pathophysiology by decreasing the ability of the TM to modulate outflow resistance.