Ubiquitin is a member of the family of low-molecular-weight heat shock proteins that serve a vital role in physiological and pathological protein turnover. It appears to be one of the proteins involved in cell alterations during aging, degenerative disorders, and age-related cognitive decline. It is not known exactly how ubiquitin alterations are related to aging disorders; however, it is possible that ubiquitin is one of the target proteins for free-radical attack. In vivo, the free radical superoxide reacts with nitric oxide to form peroxynitrite, a powerful oxidant. Peroxynitrite may react directly with proteins, lipids, and other molecules to cause damage, with ubiquitin being a possible target. In vitro reaction of peroxynitrite with ubiquitin produces two modified forms of the protein, one oxidized at methionine and the other nitrated at tyrosine, which were characterized by electrospray ionization time-of-flight mass spectrometry. The exact location of the nitrated tyrosine residue was determined by in-source collision-induced dissociation using electrospray ionization time-of-flight mass spectrometry.