Paired helical filaments (PHFs) are the structural constituents of 
neurofibrillary tangles in Alzheimer's disease and are composed of 
hyperphosphorylated forms of the microtubule-associated protein tau (PHF-tau). 
Pathological hyperphosphorylation of tau is believed to be an important 
contributor to the destabilisation of microtubules and their subsequent 
disappearance from tangle-bearing neurons in Alzheimer's disease, making 
elucidation of the mechanisms that regulate tau phosphorylation an important 
research goal. Thus, it is essential to identify, preferably by direct 
sequencing, all of the sites in PHF-tau that are phosphorylated, a task that is 
incomplete because of the difficulty to date of purifying insoluble PHF-tau to 
homogeneity and in sufficient quantities for structural analysis. Here we 
describe the solubilisation of PHF-tau followed by its purification by Mono Q 
chromatography and reversed-phase HPLC. Phosphopeptides from proteolytically 
digested PHF-tau were sequenced by nanoelectrospray mass spectrometry. We 
identified 22 phosphorylation sites in PHF-tau, including five sites not 
previously identified. The combination of our new data with previous reports 
shows that PHF-tau can be phosphorylated on at least 25 different sites.