Filamentous alpha-synuclein deposition is the defining hallmark of neurodegenerative synucleinopathies. The onset and progression of these diseases are thought to be related the formation of the alpha-synuclein filaments. We have analyzed posttranslational modifications of the filamentous alpha-synuclein in synucleinopathy brains by biochemical and protein chemical techniques. Mass spectrometric analysis revealed that deposited alpha-synuclein is highly phosphorylated at Ser129. We also found that alpha-synuclein is ubiquitinated in several synucleinopathy brains. The ubiquitination sites of soluble and filamentous alpha-synuclein were determined. These data have important implications for understanding the formation of alpha-synuclein filaments in synucleinopathy brains.