NEW PHOSPHORYLATION SITES IDENTIFIED IN HYPERPHOSPHORYLATED TAU (PAIRED HELICAL FILAMENT-TAU) FROM ALZHEIMERS-DISEASE BRAIN USING NANOELECTROSPRAY MASS-SPECTROMETRY

Paired helical filaments (PHFs) are the structural constituents of neu rofibrillary tangles in Alzheimer's disease and are composed of hyperp hosphorylated forms of the microtubule-associated protein tau (PHF-tau ). Pathological hyperphosphorylation of tau is believed to be an impor tant contributor to the destabilisation of microtubules and their subs equent disappearance from tangle-bearing neurons in Alzheimer's diseas e, making elucidation of the mechanisms that regulate tau phosphorylat ion an important research goal. Thus, it is essential to identify, pre ferably by direct sequencing, all of the sites in PHF-tau that are pho sphorylated, a task that is incomplete because of the difficulty to da te of purifying insoluble PHF-tau to homogeneity and in sufficient qua ntities for structural analysis, Here we describe the solubilisation o f PHF-tau followed by its purification by Mono Q chromatography and re versed-phase HPLC, Phosphopeptides from proteolytically digested PHF-t au were sequenced by nanoelectrospray mass spectrometry. We identified 22 phosphorylation sites in PHF-tau, including five sites not previou sly identified. The combination of our new data with previous reports shows that PHF-tau can be phosphorylated on at least 25 different site s.