Comparison of Common and Disease-Specific Post-translational Modifications of Pathological Tau Associated With a Wide Range of Tauopathies.

Brain Tissues, Preparation of Insoluble Tau, and Immunohistochemistry The cases selected for this study [three AD, three FTDP-17T (intron 10 + 16), three PSP, three CBD, three GGT, and three PiD cases] are listed in Table 1 . TABLE 1 Description of the cases used PTMs analysis. Case No. Age at death Gender Brain weight pmi Brain region Neuropathological diagnosis AD1 85 M 1146 8 h Parietal AD AD2 94 F 983 11 h Parietal AD AD3 61 M nd nd Frontal AD FTDP-17T1 55 M 1240 nd Frontal MAPT +16 FTDP-17T2 53 M 1240 nd Frontal MAPT +16 FTDP-17T3 65 F 1040 nd Frontal MAPT +16 CBD1 73 M 1200 3 h Frontal CBD CBD2 74 F 899 nd Frontal CBD CBD3 74 F nd nd Frontal CBD GGT1 74 M 1222 10 h Frontal GGT GGT2 77 F 1110 2 h Frontal GGT GGT3 76 F nd nd Frontal GGT PSP1 63 M 1435 2 h Frontal PSP PSP2 85 M 850 1 h Frontal PSP PSP3 82 M 1280 nd Frontal PSP PiD1 62 F 928 nd Frontal Pick PiD2 56 M 1150 nd Frontal Pick PiD3 nd nd nd nd Frontal Pick Sarkosyl-insoluble tau was prepared as previously described ( Zhang et al., 2020 ); this method was developed and afforded highly purified pathological tau proteins. Briefly, 0.5 g tissues were was homogenized in 20 volumes (v/w) of extraction buffer, brought to 2% sarkosyl, and incubated for 30 min. The supernatant after a 10 min spin at 20,000 g , was centrifuged at 168,000 × g for 20 min. The pellet was resuspended in a small amount of extraction buffer and centrifuged at 9,500 g for 10 min. The supernatant was diluted threefold in 50 mM Tris–HCl, pH 7.5, containing 0.15 M NaCl, 10% sucrose and 0.2% sarkosyl, and spun at 168,000 g for 20 min. For immunoblotting, the sarkosyl-insoluble pellet was resuspended in SDS sample buffer and the aliquots were subjected to SDS-PAGE on 4∼20% polyacrylamide gradient gel. Immunoblotting was performed with anti-tau C-terminus antibody T46 as described elsewhere ( Taniguchi-Watanabe et al., 2016 ). For immunohistochemistry, brain tissues were fixed by in 10% buffered formalin and embedded in paraffin, then 8-micron-thick sections were prepared and immunostained with a monoclonal antibody AT8 (Invitrogen). LC-MS/MS Analysis of Sarkosyl-Insoluble Tau Sarkosyl-insoluble fractions containing 500∼5000 ng of tau were treated with 70% formic acid for 1 h at room temp, then diluted in water and dried up. For trypsin digestion, 50 mM triethylammonium bicarbonate and 1 μg of Trypsin/Lys-C Mix (Promega) were added. Each mixture was incubated at 37°C for 20 h. After tryptic digestion, 2 μL of 100 mM DTT was added to the mixture, and incubation was continued at 100°C for 5 min. Then the sample was dried and stored at −80°C until assay. Each sample was resuspended in 0.1% formic acid and introduced into a nano-flow HPLC system, EASY-nLC 1200 (Thermo Fisher Scientific Inc., Waltham, MA, United States). A packed nano-capillary column NTCC-360/75-3-123 (0.075 mm I.D. × 125 mm L, particle diameter 3 μm, Nikkyo Technos Co., Ltd., Tokyo, Japan) was used at a flow rate of 300 nl/min with a 2–80% linear gradient of acetonitrile for 80 min. Eluted peptides were directly detected with an ion trap mass spectrometer, Q-Exactive HF (Thermo Fisher Scientific Inc., Waltham, MA, United States). For ionization, a spray voltage of 2.0 kV and a capillary temperature of 250°C was were used. The mass acquisition method consisted of one full MS survey scan with an Orbitrap resolution of 60,000, followed by an MS/MS scan of the most abundant precursor ions from the survey scan with an Orbitrap resolution of 15,000. Dynamic exclusion for the MS/MS was set to 30 s. The MS scan range of 350–1800 m/z was employed in the positive ion mode, followed by data-dependent MS/MS using the HCD operating mode on for the top 15 ions in order of abundance. The data were analyzed with Proteome Discoverer (Thermo Fisher Scientific Inc., Waltham, MA, United States), Mascot software (Matrix Science Inc., Boston, MA, United States) and Scaffold software (Proteome Software, Inc., Oregon, OR, United States). Swissprot and GenBank databases were used. Mass spectrometry data are obtained from jPOST (Japan ProteOme STandard Repository), which is certificated member of ProteomeXchange Consortium. ID number is PXD020371.

Supplementary Material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins.2020.581936/full#supplementary-material Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.