Parkinson's disease is a movement disorder that results from a loss of dopaminergic neurons in the substantia nigra. The disease is characterized by mitochondrial dysfunction, oxidative stress, and the presence of “Lewy body” inclusions enriched with aggregated forms of α-synuclein, a presynaptic protein. Although α-synuclein is modified at various sites in Lewy bodies, it is unclear how sequence-specific posttranslational modifications modulate the aggregation of the protein in oxidatively stressed neurons. To begin to address this problem, we developed an affinity pull-down/mass spectrometry method to characterize the primary structure of histidine-tagged α-synuclein isolated from catecholaminergic neurons. Using this method, we mapped posttranslational modifications of α-synuclein from untreated neurons and neurons exposed to rotenone, an inhibitor of mitochondrial complex I. Various posttranslational modifications suggestive of oxidative damage or repair were identified in a region comprising a 20-residue stretch in the C-terminal part of the protein. The results indicate that α-synuclein is subject to discrete posttranslational modifications in neurons with impaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine how specific modifications of α-synuclein contribute to neurologic disorders such as Parkinson's disease.