*2.1. p300-Mediated Acetylation of Tau*

Hyperacetylated Tau (Ac-Tau) was prepared from wt Tau using p300 HAT (see Section 3). Tau acetylation was verified by Western blot against acetyl-lysines (Figure 1B) and mass spectrometry (Figure 1C–E). We identified 15 acetylation sites (99% sequence coverage) using tandem mass spectrometry (Figure 1C; Section 3), including K148 near the Tau N-terminal domain; K163, K174, K190, K224, K234, and K240 in the P1–2 regions; K254, K280, K281, K290, and K311 in the microtubule binding region (MTBR, R1–R4); and, K375, K385, and K395 in the P3 region (Figure 1A,C). Figure 1D,E shows the representative MS/MS spectra (VQIINK280K281 and VQIVYK311, respectively). Fragmented b (red) and y (blue) ions from low energy collisions in mass spectrometer are marked. The b and y ions refer to the peaks corresponding to the prefix ions observed sequentially in the spectrum with each prefix offset from the previous by the mass of an amino acid. A 42.016 Da increase in

mass difference due to lysine acetylation is included in the sequential mass difference to construct the peptide sequence. The acetylation sites that were identified are consistent with previous reports [19–21]. Notably, K280/K281 and K311, which are in the hexapeptide aggregation motifs VQIINK280K281 and VQIVYK311, were found to be acetylated (Figure 1D,E). These motifs play critical roles in Tau interaction with negatively-charged microtubules and with polyanions such as heparin [9]. If we assume complete acetylation, we expect the theoretical pI for full-length Tau to change from 8.2 to 5.5 (Prot pi web tool; https://www.protpi.ch). Such modifications can significantly alter Tau electrostatic properties, with 50% acetylation already corresponding to a pI of 6.2. Interestingly, the observed sites of acetylation are concentrated in the positively-charged central region of Tau (Figure 1A).

**Figure 1.** Tau hyperacetylation. (**A**) Domain organization of Tau. Protein segments are color-coded to reflect their respective pIs using the provided color palette. (**B**) In vitro Tau acetylation was verified by Western blot against acetyl-lysine (left to right lanes: acetylated Tau after one- and three-day reaction incubations, and negative controls using wild-type Tau and BSA, respectively). (**C**) Tau lysine acetylation sites (red) identified by mass spectrometry. (**D**,**E**) MS/MS spectra of peptides that contain the hexapeptide aggregation motifs VQIINK280K281 and VQIVYK311, showing acetylation at K280 and K311, respectively (shown in red). The 'b' ions (shown in red) represent fragment peaks generated from the amino to carboxyl terminus. The 'y' ions (shown in blue) represent fragment peaks generated from the carboxyl to amino terminus. The suffix numbers represent the corresponding number of amino acids. See Section 3 for details.
