Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau

ABSTRACT: Abnormal changes in the neuronal microtubule-associated protein Tau, such as high phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Abnormal phosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to ~20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pm showed overall occupancies centered at 8 Pi (± 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Ph had 14 Pi (± 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Spodoptera Frugiperda

TISSUE(S): Brain

DISEASE(S): Alzheimer's Disease

SUBMITTER: Friedel Drepper  

LAB HEAD: Bettina Warscheid

PROVIDER: PXD020985 | Pride | 2020-11-20

REPOSITORIES: Pride