Alzheimer's disease (AD) is a neurodegenerative disorder characterized by brain pathology of intracellular neurofibrillary tangles (NFTs) and extracellular amyloid plaques. NFTs contain aberrantly hyperphosphorylated Tau as paired helical filaments (PHFs). Although NFs have been shown immunohistologically to be part of NFTs, there has been debate that the identity of NF proteins in NFTs is due to the cross-reactivity of phosphorylated NF antibodies with phospho-Tau. Here, we provide direct evidence on the identity of NFs in NFTs by immunochemical and mass spectrometric analysis. We have purified sarkosyl-insoluble NFTs and performed liquid chromatography/tandem mass spectrometry of NFT tryptic digests. The phosphoproteomics of NFTs clearly identified NF-M phosphopeptides SPVPKS* PVEEAK, corresponding to Ser685, and KAES* PVKEEAVAEVVTITK, corresponding to Ser736, and an NF-H phosphopeptide, EPDDAKAKEPS* KP, corresponding to Ser942. Western blotting of purified tangles with SMI31 showed a 150-kDa band corresponding to phospho-NF-M, while RT97 antibodies detected phospho-NF-H. The proteomics analysis also identified an NF-L peptide (ALYEQEIR, EAEEEKKVEGAGEEQAAAK) and another intermediate filament protein, vimentin (FADLSEAANR). Mass spectrometry revealed Tau phosphopeptides corresponding to Thr231, Ser235, Thr181, Ser184, Ser185, Thr212, Thr217, Ser396, and Ser403. And finally, phosphopeptides corresponding to MAP1B (corresponding to Ser1270, Ser1274, and Ser1779) and MAP2 (corresponding to Thr350, Ser1702, and Ser1706) were identified. In corresponding matched control preparations of PHF/NFTs, none of these phosphorylated neuronal cytoskeletal proteins were found. These studies independently demonstrate that NF proteins are an integral part of NFTs in AD brains.—Rudrabhatla, P., Jaffe, H., Pant, HC Direct evidence of phosphorylated neuronal intermediate filament proteins in neurofibrillary tangles (NFTs): phosphoproteomics of Alzheimer's NFTs.