Human brain samples
Anonymized human post-mortem tissue was obtained from the London Neurodegenerative Diseases Brain Bank and the Southwest Dementia Brain Bank, members of the Brains for Dementia Research Network, as well as the Netherlands Brain Bank (NBB), Netherlands Institute for Neuroscience, Amsterdam. Donor characteristics are described in Supplementary Tables S1 and S3. All Material has been collected from donors for whom a written informed consent for a brain autopsy and the use of the material and clinical information for research purposes had been obtained by the respective brain bank.
Transgenic mouse brain samples
Heterozygous rTg4510 mice and the corresponding wild-type (WT) line (licensed from the Mayo Clinic, Jacksonville Florida, USA) [21] were used in the study. All mice were bred for AbbVie by Charles River Laboratories (Sulzfeld, Germany). The mice were in a temperature- and humidity-controlled room with a 12:12 h dark/light cycle with ad libitum access to water and food. All animal experiments were performed in full compliance with the Principles of Laboratory Animal Care (NIH publication No. 86–23, revised 1985) in an AAALAC accredited program where veterinary care and oversight was provided to ensure appropriate animal care. All animal studies were approved by the government of Rhineland Palatinate (Landesuntersuchungsamt Koblenz) and conducted in accordance with the directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purpose, the ordinance on the protection of animals used for experimental or scientific purposes (German implementation of EU directive 2010/63; BGBl. I S. 3125, 3126), the Commission Recommendation 2007/526/EC on guidelines for the accommodation and care of animals used for experimental and other scientific purposes, the German Animal Welfare Act (BGBl. I S. 1206, 1313) amended by Art. 1 G from 17 December 2018 I 2586. Tg4510 mice express 0N4R human P301L mutant hTau under the CaMKIIα promoter. For this study, mice were culled and hippocampi and cortices were harvested from 8-, 16- and 30-week old heterozygous rTg4510 mice and 16 week old WT mice and stored at − 80 °C until use.
Brain lysate preparation
Human and mouse brain samples were homogenized in Triton lysis buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100 and protease, phosphatase, demethylase (500 μM IOX1, 2 μM Daminozide, 10 μM Paragyline Hydrochloride), deacetylase (10 μM Trichostatin A, 5 mM Nicotinamide), O-GlcNAcase (1 μM Thiamet-G) inhibitors) with a Dounce homogenizer (Carl Roth). The tubes were twice scraped over a hard surface with in-between incubation on ice for 10 min. Samples were centrifuged for 20 min at 16000x g at 4 °C, and the protein concentration in the supernatant was determined with a BCA assay (BioRad, cat. No. 5000112).
Triton-insoluble pellets from mouse brain were resuspended in sample buffer (65 mM Tris-HCl, 5% Glycerol, 1% SDS) and boiled for 15 min at 95 °C, followed by centrifugation for 25 min at 16000x g, 4 °C. The supernatant containing Triton-insoluble Tau was used for subsequent Western blot analyses.
Sarkosyl extraction of human brain samples
Sarkosyl extraction was performed as described previously ( [22, 23], Suppl. Fig. S3). Briefly, brain tissue was homogenized in TBS buffer (50 mM Tris pH 7.4, 150 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 0.5 mM MgSO4, complete protease inhibitor with EDTA (Roche), PhosSTOP phosphatase inhibitor (Roche)) with a cooled bead ruptor (FastPrep-24, MP Biomedicals) at 5 μl/mg tissue. Homogenates were centrifuged at 270000 x g and 4 °C for 20 min to obtain the supernatant S1 and pellet P1. S1 was flash frozen and stored at − 80 °C until use. P1 was suspended in the original volume of salt/sucrose buffer (10 mM Tris/HCl pH 7.4, 1 mM EGTA, 0.8 M NaCl, 10% sucrose, complete protease inhibitor with EDTA (Roche) and PhosSTOP phosphatase inhibitor (Roche)) at room temperature (RT) with a bead ruptor (Omni International, Inc). Samples were centrifuged at 27000x g and 4 °C for 20 min, the resulting supernatant S2 was adjusted to a final concentration of 1% sarkosyl and incubated for 1.5 h at RT with 750 rpm shaking. Samples were centrifuged at 150000x g and 4 °C for 45 min, the sarkosyl-insoluble pellet P3 was washed once in TBS (25 mM Tris pH 7.4, 3 mM KCl, 140 mM NaCl) and resuspended in TBS by vortexing and four sonication pulses (2 s each) with a probe sonicator (Sonoplus, Bandelin) at 35% amplitude. For ELISA, the extracts were mixed with an equal volume of 10 μl 2x Laemmli buffer (Bio-Rad), heated for 5 min at 98 °C, flash frozen and stored at − 80 °C until use.
Immunoprecipitation of brain samples and tau PTM analysis by LC-MS/MS
250 μg protein from each human brain sample lysed in Triton lysis buffer was used for immunoprecipitation (IP) with a combination of Tau 12 (Biolegend, cat. no. 806501), Tau 5 (Abcam, cat. no. ab80579) and HT7 (Thermo Fisher, cat. no. MN1000) as described previously [20]. Briefly, immunoprecipitated samples were eluted with 50 mM glycine HCl pH 2.8, separated by SDS-PAGE and Coomassie-stained bands were excised. Samples were subjected to either an in-gel tryptic- or to an in-gel AspN digest, and peptides were separated using nanoAcquity UPLC (Waters) with a nanoAcquity trapping (nanoAcquity Symmetry C18, 5 μm, 180 μm × 20 mm) and analytical column (nanoAcquity BEH C18, 1.7 μm, 75 μm × 200 mm), which was coupled to an LTQ Orbitrap Velos Pro (Thermo Fisher) using the Proxeon nanospray source as previously described [20].
Acquired LC-MS/MS data were processed using IsobarQuant [24] and Mascot (v2.2.07) with a reversed Uniprot Homo sapiens database (UP000005640) including common contaminants. The following modifications were taken into account: Carbamidomethyl (C) (fixed modification), as well as Methyl (K), Dimethyl (K), Acetyl (K), Acetyl (N-term), Phospho (ST), Phospho (Y) and Oxidation (M) (variable modifications). The mass error tolerance for full scan MS spectra was set to 10 ppm and for MS/MS spectra to 0.5 Da. A maximum of 2 missed cleavages were allowed. A minimum of 2 unique peptides with a peptide length of at least seven amino acids and a false discovery rate below 0.01 were required on the peptide and protein level. Peptides with a MASCOT Score ≥ 20 were determined as reliable detected peptides [25]. The data have been deposited to the ProteomeXchange Consortium via the PRIDE [26] partner repository with the dataset identifier PXD017065.
Methyl- tau antibodies
Immunization and antibody purification were performed by Innovagen (Lund, Sweden). Antibodies were raised in rabbits against the respective methylated peptides indicated in Suppl. Fig. S1. The animals were subjected to a total of three booster injections, with the final immune serum harvest 2 months after the first immunization. Immune sera were affinity purified against the appropriate methyl-peptide, and the purified antibodies were stored in aliquots in PBS at − 80 °C until use.
Recombinant tau purification
Full length Tau 2N4R was expressed and purified as described previously [20]. Briefly, E. coli BL21(DE3) cells (Sigma, cat. no. CMC0014) transformed with a pET19n vector containing 2N4R Tau were induced by the addition of 1 mM IPTG and harvested by centrifugation. Cleared lysates in 50 mM Na-phosphate pH 7.0, 1 mM EGTA, 1 mM DTT, cOmplete protease inhibitors (Roche), benzonase (Merck) and 10 μg/ml lysozyme (Sigma) were boiled for 20 min at 100 °C. Supernatants were loaded onto a combination of a HiTrap Q and a HiTrap SP column (GE Healthcare) and eluted in a gradient to running buffer containing 300 mM Nacl. tau-containing fractions were further purified with a HiLoad 16/600 Superdex 75 pg size exclusion chromatography column (GE Healthcare). Fractions were analyzed by SDS-PAGE, pooled according to purity, flash-frozen in liquid nitrogen and stored at − 80 °C.
In vitro methylation of recombinant tau
100 μg of recombinant Tau 2N4R were incubated for 2 h at RT in 100 mM sodium citrate pH 6 (Sigma, cat. no. W302600), 100 mM sodium cyanoborohydride (Sigma, cat. no. 156159) and 20 mM formaldehyde (Fisher Scientific, cat. no. 28908) with a final volume of 100 μl. 10 μl of 550 mM glycine (Roth, cat. no. 3187) were added, and Zeba™ Spin desalting columns (Thermo Fisher, cat. no. 87767) were used according to manufacturer’s instructions to remove excessive sodium cyanoborohydride.
In vitro dephosphorylation with lambda phosphatase
5 μl aliquots of the respective brain fractions were mixed with 1 μl lambda phosphatase (New England Biolabs, cat. no. P0753), 10x buffer and MnCl2 were added according to manufacturer’s instructions in a total volume of 10 μl. Control reactions were set up without addition of the enzyme. After incubation for 30 min at 30 °C, 2.5 μl of 4x Laemmli sample buffer were added, and samples were processed as described below for immunoblotting.
Immunoblotting
To generate dot blots for testing the specificity of methyl-Tau antibodies, 3 μg methylated peptides, bare peptides, in vitro methylated full-length Tau (2N4R) and recombinant Tau (2N4R) were spotted onto nitrocellulose membranes.
For Western blots, 25 μg total protein were boiled at 95 °C for 5 min in sample buffer, separated on a 10% SDS-PAGE and immunoblotted onto PVDF membranes.
The membranes were blocked with blocking buffer (5% BSA in TBST (Roth)) and incubated with primary antibodies overnight at 4 °C: Tau 12, mouse, 1:500, (Biolegend, cat. no. 806501); Tau 5, mouse, 1:1000, (Abcam, cat. no. ab80579); meK130, rabbit, 1:500, (INNOVAGEN); meK132, rabbit, 1:1000 (INNOVAGEN); meK343, rabbit, 1:1000 (INNOVAGEN); meK353, 1:200, (INNOVAGEN); meK438, rabbit, 1:1000 (INNOVAGEN); Actin, rabbit 1:1000, (Cell Signaling Technology, cat. no. 4910); SETD7, mouse, 1:1000, (Thermo Fisher, cat. no. 730055); SETD7, rabbit, 1:1000, (Invitrogen, cat. no. MA5–35782); Hsp90, mouse, 1:1000, (Millipore, cat. no. 05–594); HDAC2, rabbit, 1:1000, (Cell Signaling Technology, cat. no. 2545S). Antibodies were diluted in 5% BSA in 1X TBST (1X TBS, 0.05% Tween-20). The next day, secondary antibody incubations (1:20000, IRDye Donkey anti-mouse 800 and IRDye Donkey anti-rabbit 680) were performed in blocking solution for 1 hour at RT. The membranes were then washed three times with 1X TBST, once with 1X TBS and imaged on a Li-Cor Odyssey CLx scanner. Alternatively, AzureSpectra antibodies (IRDye goat anti-mouse 800, cat. no. 201206–58, 1:5000, and IRDye goat anti-rabbit 650, cat. no. 180309–72,1:5000) were used and imaged using ChemiDoc Touch MP (Bio-Rad) and analysed via ImageLab6.
For weak signals, anti-rabbit HRP- linked antibody was used (Cell Signaling Technology, 1:5000) in blocking buffer, with incubation for 1 hour at RT. The membranes were then washed three times with 1X TBST, once with 1X TBS. ECL solution clarity Max (Bio-Rad, cat. no. 1705062) was added to the membrane and chemiluminescent signals were detected on a Fusion FX7 (Vilber).
Electrochemiluminescence ELISA
To quantify methylated Tau in mouse brain samples and SHSY5Y cells, Gold Streptavidin small-spot 96-well plates (Meso Scale Discovery, cat. no. L45SA) were blocked with 5% (w/v) Blocker A solution in Tris wash buffer for 1 h at RT on a plate shaker. Plates were washed three times with Tris wash buffer and coated with 1 μg of biotinylated antibody diluted in 25 μL of 1% Blocker A solution for 1 h at RT on a plate shaker. Antibody biotinylation was performed according to manufacturer’s instructions (EZ-Link Sulfo-NHS-Biotin, Thermo Scientific, cat. no. 21217), after BSA removal with the Melon Gel IgG Purification Kit (Thermo Scientific, cat. no 45212) if necessary. For mouse brain samples, biotin Tau5 (Abcam) was used as a total Tau antibody. For SHYSY5Y cells, biotin HT7 (Thermo Fisher, cat. no. MN1000) was used. Antibody-coated plates were washed three times with Tris wash buffer, 5 μg of protein lysates (diluted to 50 μl with TBS) were added and incubated for 1 h at RT on a plate shaker. Plates were washed three times with Tris wash buffer and incubated for 1 h at RT on a plate shaker with 25 μl of 1 μg/ml detection antibody (for mouse samples: HT7; for SHSY5Y samples: Tau 12, both labeled with MSD Sulfo-Tag-NHS-Ester, Meso Scale Discovery) diluted in 1% Blocker A solution. Plates were washed three times with Tris wash buffer and 150 μl of 2X Read Buffer were added 5 minutes before the signal was measured on a MESO QuickPlex SQ 120 (Meso Scale Discovery).
Colorimetric ELISA
ELISA plates (Maxi Sorp, Thermo Scientific) were coated over night at 4 °C with 100 μl capture antibody per well (2 μg/ml Tau-12 (Biolegend, cat. no 806502), meK130 or meK132, AT8 (Invitrogen, cat. no. MN1020) or AT100 (Invitrogen, cat. no. MN1060) antibody in 20 mM NaH2PO4 pH 7.4, 140 mM NaCl, 20% glycerol). The next day, plates were washed 3x with 250 μl/well wash buffer (20 mM NaH2PO4 pH 7.4, 140 mM NaCl, 0.05% Tween 20) and blocked for 1.5 h at RT with 250 μl/well blocking buffer (20 mM NaH2PO4 pH 7.4, 140 mM NaCl, 0.05% Tween 20, 20% glycerol, 2% BSA). Samples were diluted in assay buffer (20 mM NaH2PO4 pH 7.4, 140 mM NaCl, 0.05% Tween 20, 0.1% BSA) to fall within the experimentally validated linear range of the assay. Plates were washed 3x with 250 μl/well wash buffer and 100 μl/well samples were added. All incubation steps of meK132 and meK130 ELISA plates were performed on an orbital shaker at 650 rpm. After 2 h incubation at RT, plates were washed 3x with 250 μl/well wash buffer. For plates with Tau 12 as capture antibody, 100 μl/well of 0.1 mg/ml Biotin-HT7 antibody (Invitrogen, MN1000B) in assay buffer were added. For plates with meK130, meK132, AT8 or AT100 as capture antibodies, 100 μl/well of 0.1 mg/ml Biotin-Tau 12 (Biolegend) were added. Plates were incubated for 1 h at RT, washed 3x with 250 μl/well wash buffer, and 100 μl/well of Pierce Streptavidin poly-HRP conjugate were added (Thermo Scientific, diluted 1:10000 in assay buffer). The plates were incubated for 1 h at RT, washed 3x with 250 μl/well wash buffer, and 100 μl/well TMB ELISA substrate were added (KEM EN TEC Diagnostics). After 10 min incubation at RT in the dark, the reaction was stopped by adding 100 μl/well 0.18 M H2SO4, and the absorbance was read at 450 nm.
Culture and transfection of cell lines
HEK 293 T cells were cultured at 37 °C in 5% CO2 in DMEM+ GlutaMax (Thermo Fisher, cat. no. 10566016) with 10% fetal bovine serum (Sigma, cat. no. F7524-500 ml) and 1% penicillin/streptomycin (Fisher Scientific, cat. no. 15307583). Cells were used for transfections between passage 10–20. Transfection was performed at a cell confluency of 60–70% using JetPrime reagent (PolyPlus) according to manufacturer’s instructions. For each construct, 1 μg DNA (WT 2N4R Tau, K130R 2N4R Tau, K132R 2N4R Tau, K343R 2N4R Tau, K353R 2N4R Tau and K438R 2N4R Tau) was used. Tau constructs contained an N-terminal HA tag and were cloned into the pcDNA3 vector, mutants were generated by site-directed mutagenesis (Q5 site directed mutagenesis, New England BioLabs, cat. no. E0552S) and verified by sequencing. Cells were transfected and incubated for 24 h before they were harvested by scraping, centrifuged for 5 min at 2000x g at 4 °C, washed once with ice-cold PBS and centrifuged again for 5 min at 2000x g at 4 °C. Samples were stored at − 20 °C until further use.
SHSY5Y neuroblastoma cells stably expressing GFP-0N4R Tau (Innoprot, cat. no. P30722-4R) were cultured at 37 °C in 5% CO2 in RPMI 1640 + GlutaMax media (Thermo Fisher, cat. no. 61870044) with 10% fetal bovine serum and 250 μg/G418 (Fisher Scientific, cat. no. 10131035). Cells were used for experiments between passage 3–20. 5 μM (R)-PFI-2 (Sigma cat. no. SML1408) was added to the cells at a confluency of 50%, followed by an incubation for 16 h. Cells were harvested by scraping, centrifuged for 5 min at 2000x g at 4 °C, washed once with ice-cold PBS and centrifuged for 5 min at 2000x g at 4 °C. Samples were stored at − 20 °C until further use.
Treatment with PKMT inhibitors and cell viability test
Compounds described as selective PKMT inhibitors by the Structural Genomics Consortium (SGC, [27]) were sourced as outlined in Suppl. Table S2, stock solutions were prepared in DMSO and stored at − 20 °C until use. SHSY5Y cells were seeded into 96well plates and treated with PKMT inhibitors in the medium at the concentrations indicated in Suppl. Table S2 for 24 h. Cell titer blue reagent (Promega) was added according to manufacturer’s instructions, incubated with the cells for 3 h and fluorescence was measured with an excitation at 560 nm and emission at 590 nm. Cells were washed once with PBS, lysed and processed for ELISA as described above.
hiPSC neuron culture
Neurogenin 2 (NGN2)- induced neurons were differentiated as previously described with minor modifications [28]. A doxycycline inducible NGN2 expression cassette was stable integrated in the AAV1 locus using TALEN technology by Bioneer (Denmark). hiPSCs were split in a concentration of 100,000 cells/cm2 on matrigel (BD) coated plates in mTesR (Stem Cell Technologies). At day 1 after splitting, the medium was changed to N2/B27 medium (50% DMEM/F12, 50% Neurobasal, 1:200 N2, 1:100 B27, 1% PenStrep, 0.5 mM Non-essential amino acids, (all Invitrogen), 50 μM ß-mercaptoethanol (Gibco), 2.5 μg/ml insulin and 1 mM sodium pyruvate (both Sigma)) with 2 μg/ml doxycycline (Sigma). The medium was changed daily. On day 4, cells were split with accutase (Invitrogen) and re-seeded in a density of 200,000 cells/cm2 in N2/B27 medium with doxycycline and 10 μM Rock inhibitor Y- 27632 (Selleckchem) on matrigel coated plates in the final format. N2/B27 with doxycycline was changed daily until day 7. On day 8 the medium was switched to final maturation medium (FMM; N2/B27 with 20 ng/ml BDNF, 10 ng/ml GDNF (both Peprotech), 1 mM dibutyryl-cAMP (Sigma) and 200 μM ascorbic acid (Sigma)). The medium was changed every third day until cells were used for treatment with (R)PFI-2 at day 21. N2/B27 medium with DMSO or 5 μM (R)-PFI-2 [29] were added and refreshed once after 24 h. 48 h after initial treatment, cells were detached with ice-cold PBS and centrifuged for 5 min at 2000x g at 4 °C. Samples were stored at − 20 °C until further use.
Proximity ligation assay (PLA)
Specific primary antibodies against Tau (Tau12, mouse, 1:500, Biolegend, cat. no. 806501) and total mono-methyl Lysine (meK, rabbit, 1:500, Cell Signaling, cat. no. 14679S) were used to detect methylated Tau in hiPSC-derived neurons. A combination of the Tau12 antibody with an additional total Tau antibody (Tau, rabbit, 1:500, Dako, cat. no. A0024) served as positive control. Negative controls consisted of applying only one primary antibody against Tau (Tau12) and of omitting the primary antibodies (not shown). PLA was performed in MAPT wildtype and MAPT knockout hiPSC-derived neurons. Duolink In Situ PLA red mouse/rabbit kit was purchased from Sigma Aldrich (DUO92002, DUO92004, DUO82049, DUO92008; Sigma-Aldrich, St. Louis, Missouri, USA). This kit includes mouse and rabbit secondary antibodies with probes, blocking solution, wash buffers A and B, amplification solution, ligase solution and detection reagent. PLA was performed according to manufacturer’s instructions.
For PLA analysis, images were acquired using a Leica LSM800 confocal laser-scanning microscope with a 25× oil immersion objective (Leica, Wetzlar, Germany). Thickness of single optical sections was 0.83 μm in stacks of ~ 14 μm total depth. Individual images were composed of 6 tiles (3 × 2 tiles in x- and y-direction, respectively).
For quantification of methylated Tau in hiPSC-derived neurons, 9 images per condition were analyzed for each biological replicate. Three technical and biological replicates for each condition were applied. Nuclear meK-Tau12 PLA signal in hiPSC-derived neurons was measured using ImageJ (Fiji). The integrated density of PLA probe signal in nuclei was quantified in a mask created of Hoechst-labeled nuclei.
SETD 7 Knock down
shRNA sequences against SETD7 were designed using an online tool (www.biosetta.com) and synthesized (Eurofins). Oligonucleotide sequences were as follows:
shRNA-1:
top: 5′ – ACCGGGCCAGGGAGTTTACACTTAGTTAATATTCATAGCTAAGTGTAAACTCCCTGGCTTTT – 3′.
bottom: 5′ – CGAAAAAAGCCAGGGAGTTTACACTTAGCTATGAATATTAACTAAGTGTAAACTCCCTGGCC – 3′.
shRNA-2:
top: 5′ – ACCGGGGGAGTTTACACTTACGAAGTTAATATTCATAGCTTCGTAAGTGTAAACTCCCTTTT – 3′.
bottom: 5′ – CGAAAAAAGGGAGTTTACACTTACGAAGCTATGAATATTAACTTCGTAAGTGTAAACTCCCC – 3′.
shRNA oligonucleotides were cloned into the DECIPHER pRSI12-U6-(sh)-HTS4-UbiC-TagRFP-2A-Puro lentiviral expression vector (Cellecta, cat. no. DVLIB-PS) following manufacturer’s instructions and then transformed into OneShot Top10 E.coli (Thermo Fisher, cat. no. C404003). Positives clones were verified by sequencing. 1 μg of two different shRNAs were packaged with lentiviral plasmids (MD2G, psPAX2) and transfected into HEK cells using JetPrime reagent (PolyPlus) according to manufacturer’s instructions. Media was collected 48 h after transfection and concentrated using Lenti-X™ concentrator (Takara Bio Europe SAS, cat. no. 631231) according to manufacturer’s instructions. Viral pellets were resuspended in 300 μl of DMEM and stored at − 80 °C.
SHSY5Y cells were seeded at 20% confluency in a 12 well plate before adding 10 μL lentivirus with shRNA. Cells were cultivated for 7 days with a media change on every third day. Cells were harvested by scraping, centrifuged for 5 min at 2000x g at 4 °C, washed once with ice-cold PBS and re-centrifuged for 5 min at 2000x g at 4 °C. Samples were stored at -80 °C until further use.
The knock down efficiency was analyzed using qPCR: RNA was extracted from the cell pellets using RNeasy Plus Mini Kit (QIAGEN, cat. no. 74134) according to the manufacturer’s protocol. A reverse transcription reaction was carried out to synthesize 250 ng cDNA using the High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific, cat. no. 10400745), and qPCR was performed with the GoTaq® qPCR Master Mix (Promega, cat. no. A6001) following the manufacturer’s protocol on a Quant Studio 3 real-time PCR machine (Thermo Fisher).
Subcellular fractionation of mouse brain and cultured neurons
For subcellular fractionation, hiPSC-derived neurons were washed off the culture plate in ice-cold PBS, pelleted and fractionated using the Pierce Subcellular fractionation kit for cultured cells (Thermo Fisher) according to manufacturer’s instructions. Mouse brain samples were fractionated using the Pierce Subcellular fractionation kit for tissues (Thermo Fisher) according to manufacturer’s instructions. For analysis, 2–10 μl of each fraction were run on an SDS-PAGE and subjected to Western blotting as described above.
MS proteomics analysis of subcellular fractions
Each fraction sample (100 μl) was reduced by 1 μl 1 M Dithiothreitol (DTT) at 60 °C for 30 min and alkylated by 4 μl 1 M iodoacetamide (IAM) in the darkroom for 30 min. Then proteins were precipitated with 7 volumes of chilled acetone at − 20 °C overnight. Proteins were then pelleted by centrifugation at 14,000x g for 10 min at 4 °C. Supernatant was carefully removed, and pellets were rinsed with 500 μl methanol. Protein pellets were resuspended in 80 μL of 100 mM ammonium bicarbonate and digested by addition of trypsin/Lys-C (Promega, Madison, WI) at a protein-to-total enzyme ratio of 50:1 at overnight. Trifluoroacetic acid (TFA) was added to a final concentration of 1% (v/v) to quench the digestion. Peptide concentration was determined by measuring optical density (OD) at 280 nm using Trinean DropSense 96 (PerkinElmer, Waltham, MA). Peptide concentration was then adjusted to 1 μg/μl using 2% methanol, 0.1% TFA in water and transferred to sample vial for LC-MS/MS analysis.
Peptide samples were analyzed using a Thermo Scientific EASY-nLC 1000 system coupled to a Orbitrap Fusion Lumos Mass Spectrometer (Thermo Scientific, Waltham, MA). The LC system was equipped with a PepMapTM RSLC C18 column (75 μm × 75 cm, 2 μm, Thermo Scientific, Waltham, MA) maintained at 50 °C. Solvent A consisted of 0.1% formic acid in water, and solvent B consisted of 0.1% formic acid in 90% acetonitrile. A 2 μg peptide sample was loaded onto trap with 5% acetonitrile at 10 μL/min flow rate for 5 min and then analyzed at 200 /min with LC separation gradient: 5–15% B for 5 min, 15–30% B for 170 min, 30–65% for 20 min, 65–95% B for 11 min. Data collection was operated in a 3-s cycle using the data-dependent top-speed mode. The MS1 survey scan (m/z 400–1500) was at a resolution of 240,000 (FWHM@m/z = 200), with automated gain control (AGC) target of 400,000 and a maximum injection time of 50 ms. Precursors were fragmented in HCD activation mode at a normalized collision energy of 30% and the dynamic exclusion was set with 45 s. Precursors were filtered by quadrupole using an isolation window of 1.2 amu. The MS2 spectra were collected at a resolution of 15,000 in the Orbitrap, with an AGC target of 50,000 and a maximum injection time of 50 ms.
For data processing, raw files were searched against the Uniprot-SwissProt Homo sapiens protein database containing 20,244 entries with Proteome Discoverer version 2.0 (released in July, 2015) for protein identification. The search engine Sequest HT was used and the search parameters were set to 20 ppm tolerance for precursor ion mass and 0.02 Da for fragment ion mass. Two missed cleavages were permitted for fully tryptic peptides. Carbamidomethylation of cysteine was set as a static modification, and a dynamic modification was defined as oxidation at methionine and acetylation at the N-terminal. Target FDR (strict) for decoy database search was set to 0.01 in Percolator.
Proteins and the number of unique peptides identified in each fraction are listed in Supplementary Table S6. Proteins annotated as PKMTs by the HUGO gene nomenclature committee (HGNC, www.genenames.org) were identified in each fraction and Venn diagrams were generated using the venn.diagram function in R.
Data analysis
Immunoblotting data were analyzed using Image Studio Lite (Li-cor Biosciences) and statistical analysis was performed with GraphPad Prism 7 (GraphPad Software) using the test noted within the respective figure legend. Full blots used for quantification are shown in Suppl. File S12.
Bioinformatic analysis of public RNASeq data sets
The details of the Mount Sinai Brain Bank study (MSBB) and Mayo study sample collection, RNA extraction, library preparation and sequencing were described in previous publications [30, 31]. For the analysis presented here, the reprocessed and realigned counts as described in a previous publication [32] were used. In brief, FASTQ files were aligned to GENCODE24 (GRCh38) using STAR [33]. The counts for Mayo (syn8690904, syn8690799) and MSBB (syn8691099) were downloaded from the AMP-AD knowledge portal (https://adknowledgeportal.org). For Mayo, 33 samples were excluded as described in [33] and 523 samples of two brain regions were used for the analysis. For MSBB 753 samples of 4 brain regions were used for the analysis. Genes with 1 CPM in fewer than 10 samples were removed. In both studies, differential gene expression was calculated with Limma voom [34] per brain region adjusted for RNA integrity value (RIN), age at death, post-mortem interval (PMI) and gender, in addition mayo for study center and flow cell and MSBB for race. For Mayo, the group comparisons for AD/controls/pathological aging and Braak stage V-VI/Braak stage III-IV/Braak stage 0-II were calculated, and pair-wise contrasts extracted. For MSBB the outcome groups AD/controls, Braak stage V-VI/Braak stage III-IV/Braak stage 0-II and demented (CDR 3–5)/MCI (CDR 1–2)/non dementia (CDR 0–0.5) were analyzed. Quality control, differential gene expression and plotting was performed in R [35].