*3.5. Mass Spectrometry of Acetylated Tau*

Ac-Tau sample was boiled in 30 μL of 1× NuPAGE LDS sample buffer (Invitrogen) and subjected to SDS-PAGE (NuPAGE 10% Bis-Tris gel, Invitrogen) then visualized with Coomassie Brilliant blue-stain. The SDS-PAGE gel containing the band corresponding to Tau was excised, destained, and subjected to in-gel digestion using 100 ng trypsin (#T9600, GenDepot). The digested peptides were resuspended in 10 μL of 0.1% formic acid and subjected to a nanoHPLC-MS/MS system with an EASY-nLC 1200 coupled to Fusion Tribrid Orbitrap Lumos mass spectrometer (Thermo Fisher, Waltham, MA, USA). The peptides were loaded onto a Reprosil-Pur Basic C18 (1.9 μm, Dr. Maisch

GmbH, Ammerbuch-Entringen, Germany) pre-column of 2 cm × 100 μm size. The pre-column was switched in-line with an in-housed 50 mm × 150 μm analytical column packed with Reprosil-Pur Basic C18 equilibrated in 0.1% formic acid. The peptides were eluted using a 45-min discontinuous gradient of 4–28% acetonitrile/0.1% formic acid at a flow rate of 750 nL/min. The eluted peptides were directly electro-sprayed into mass spectrometer operated in the data-dependent acquisition mode acquiring fragmentation spectra of the top 30 strongest ions under direct control of Xcalibur software (4.0; Thermo Fisher). Parent MS spectrum was acquired in the Orbitrap with full MS range of 300–1400 *m*/*z* in the resolution of 120,000. CID fragmented MS/MS spectrum was acquired in ion-trap with rapid scan mode. Obtained MS/MS spectra were searched against the target-decoy human refseq database (June 2015 release, containing 73,637 entries) in Proteome Discoverer 1.4 interface (Thermo Fisher) with the Mascot algorithm (Mascot 2.4, Matrix Science, London, UK). Variable modifications of lysine and arginine acetylation, methionine oxidation, and N-terminal acetylation were allowed. The precursor mass tolerance was confined within 20 ppm with fragment mass tolerance of 0.5 Da and with a maximum of two missed cleavages allowed. The peptides identified in the Mascot results file were validated with a 5% false discover rate (FDR) and subjected to manual verification to confirm lysine acetylation.
