Session: Session 3

Studying multi-phosphosites peptides with TrypsinN: Application to Alzheimer disease

The accumulation of aggregated Tau in brain is a common pathological feature of several neurodegenerative diseases. There is increasing evidence that tauopathies can spread between cells or connected regions of the brain. Recent advances show that in addition to the classical pathological oligomers, Tau condenses into droplets, giving it new characteristics whose impact on propagation (Boyko et al., 2022). This droplet formation is increased by different environmental factor such as phosphorylation. Our team recently focus on the AT8 phospho-epitope (ANR grants 2021), which consists of phosphorylation S202, T205 and sometimes S208 or S199. Some phosphorylations on AT8 are presumed responsible for the initial steps driving to seeding and droplets formations. Analyzing Phospho-Tau by mass spectrometry to quantify all the phosphosites is not an easy task, especially for the AT8 peptide that contains seven phosphosites but also three prolines with their N-terminal bond more labile among fragmentation. Surprisingly, a survey on phosphoproteomic studies on AD didn’t often show S208 phosphorylation that was validated by NMR (Gandhi et al., 2015).

 To sort out this problem we decide to analyze P-Tau focusing on AT8 peptides with a targeted method. Using TrypsinN (a protease that cut the N-terminal part of lysine and arginine) we improve our method and analyze different mutants of Tau to better understand the function of AT8 phosphorylations in Tau seeding. On the one hand we study the fragmentation pattern obtained with these “non tryptic”peptides, and on the other we compare the phosphorylation of AT8 obtained by Maxquant (Cox et al., 2008) between Trypsin or Trypsin N digestion with different mutants of TAU phosphorylated in vitro with mouse brain lysates.

With trypsinN we were able to quantify S208 on AT8 peptide after phosphorylation of the protein Tau with mouse brain lysates. Trypsin could help in quantifying specific phosphorylations on multi-phosphosites.