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SunMass SpectrometryBottom-up proteomicsData-independent acquisitionAlzheimer's DiseaseNeuronAlzheimer’s diseaseProteomicshttps://www.ebi.ac.uk/pride/archive/projects/PXD075357BrainWhole proteome samples were produced by lysing cell pellets with cold RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with protease inhibitor (Roche, 11873580001), PMSF (Sigma-Aldrich, 93482), and PhosSTOP (Roche, 4906837001). The lysates were rotated at 4˚C for 30 min, sonicated in a cup horn sonicator (Branson) for 90 sec at 40% amplitude, then cleared via centrifugation at 21,000 × g for 15 min at 4˚C. Supernatants were transferred to a new tube and protein concentrations were measured with Bio-Rad Protein Assay (Bio-Rad). Both whole proteome samples and IP eluates were reduced using 200 mM TCEP (Thermo Fisher Scientific, 77720) incubated at room temperature for 30 min, then alkylated with 375 mM iodoacetamide incubated at room temperature for 30 min, protected from light. Sample proteins were precipitated and washed 3 times with a precipitation solution (50% acetone, 49.9% ethanol, 0.1% acetic acid). The protein pellets were resuspended in 8 M Urea, which was then diluted to 2 M Urea by adding NaCl/Tris solution (50 mM Tris pH 8.0, 150 mM NaCl). The samples were digested with 500 ng of mass-spectrometry grade Trypsin Gold (Promega, V5280) and nutated overnight at 37˚C. The digestion was quenched by adding equal volume of 4% formic acid. The peptide samples were desalted by either loading onto Evotips according to the manufacturer’s protocol (Evosep, EV2011), or with lab-made C18 solid-phase extraction spin columns. Briefly, C18 sorbent from a Sep-Pak 200 mg C18 (Waters, WAT054945) was distributed into empty micro-spin columns (Thermo, 89879), which were conditioned and equilibrated with 80% acetonitrile in 0.1% acetic acid and 0.1% TFA, respectively by spinning at 1000 × g for 1 min each step. The samples were added to the spin columns, washed with 0.1% acetic acid, eluted with 80% acetonitrile in 0.1% acetic acid and dried in a Speed-Vac. Immediately before mass spectrometry analysis, the dried samples were resuspended in 0.1% TFA. Samples were analyzed with either a nanoElute2 (Bruker) or Evosep One (Evosep) liquid chromatography system coupled to a timsTOF HT mass spectrometer (Bruker). For the nanoElute2, a two-column separation method was employed, including a 5 mm PepMap Neo trap Cartridge (Thermo Fisher Scientific, 174500) and an in-house 10 cm by 100 µm analytical column packed with 1.9 µm C18 beads (Dr. Maisch, r119.aq.0001). Peptide separation for the nanoElute2 was performed over a 25 minute elution gradient consisting of solvent A (0.1% formic acid in water) and steadily increasing concentration of solvent B (0.1% formic acid in acetonitrile) from 2% to 35% B. For the Evosep One, an in-house 8 cm by 150 µm capillary column packed with 1.9 µm C18 beads was used with no trap column. The standard Evosep 60 SPD method was used for peptide separation. Sample acquisition was performed in data-independent acquisition mode with parallel accumulation-serial fragmentation (dia-PASEF), employing 12 cycles with estimated cycle time of 1.7 seconds. In total, 34 mass windows of 25 Da width and 2 mobility windows each were acquired with a ramp time of 75 ms to cover a mass range from 350 to 1250 Da and a mobility range from 0.64 1/K0 [Vs/cm-2] to 1.37 1/K0 [Vs/cm-2]. Collision energy was set as a function of ion mobility to follow a straight line from 20 eV at 0.6 1/K0 [Vs/cm-2] to 59 eV at 1.6 1/K0 [Vs/cm-2]. Prior to running the samples, the elution voltage calibration was confirmed, and if needed, calibrated for 1/K0 ratios using three ions from ESI-L Tuning Mix (Agilent, G1969-85000) (m/z 622, 922, 1222) in the timsControl software (Bruker).PrideNot availableRaw mass spectrometry data acquired in data-independent acquisition (DIA) mode were processed using DIA-NN (version 1.8.1). A reviewed UniProt human protein sequence database (Swiss-Prot, November 2023 release; taxonomy ID 9606) was used for all searches. Spectral libraries generated as described above were used where applicable. In experiments in which the bait protein was not detected using the specific spectral library—owing to insufficient or absent representation of bait-derived peptides—DIA-NN’s library-free search mode was employed. This strategy enabled unbiased peptide identification directly from DIA data and was essential for robust bait detection and quantification in the absence of an appropriate project-specific spectral library. For DIA-NN searches, up to three missed cleavages and a maximum of two variable modifications per peptide were allowed. Carbamidomethylation of cysteine residues and oxidation of methionine were specified as fixed modifications. The fragment ion m/z range was set from 100 to 1800. Match-between-runs (MBR) was enabled, and peptide quantification was performed using the “Any LC (high accuracy)” strategy.ProteomicsHaiyuan YuDepartment of Computational Biology Weill Institute for Cell and Molecular Biology Center for Innovative Proteomics Cornell UniversityPARTIALHomo Sapiens (human)ys795@cornell.eduNot availableCornell UniversityUnited StatesfalseInteractome mapping in human excitatory neurons reveals novel drivers in Alzheimer’s diseaseAlzheimer’s disease (AD) is an irreversible neurodegenerative disease defined by its molecular hallmarks - amyloid beta peptide plaques and neurofibrillary Tau tangles. Despite significant progress that has been made in uncovering a large number of genetic risk factors through extensive genomic sequencing and genetic studies, the molecular mechanisms driving AD-associated pathology and cognitive decline remain poorly understood. Therefore, alongside the identification of more risk genes, it is also paramount to study how these genes function and influence each other within the cellular pathways and overall molecular networks in AD-relevant brain cell types. However, current human protein-protein interactome datasets were all generated in either yeast or generic human cell lines. Consequently, many important neuronal interactions, especially neuron-specific ones, have yet been discovered. To address this critical gap, we developed a highly scalable, high-quality interactome mapping pipeline in human excitatory neurons derived from induced pluripotent stem cells (iPSC), and generated a comprehensive, neuron-specific interactome map, named ADNeuronNet, for key AD risk genes. ADNeuronNet consists of 1,767 high-confidence interactions among 1,189 proteins and is the only dataset enriched with neuron-specific genes when compared to known protein interactions, including previous large-scale interactome maps, for the same baits in the literature. Within ADNeuronNet, we identified 1,375 novel interactions, many of which are likely neuron specific. For example, we identified a neuron-specific interactor, RIN2, for major AD risk factor BIN1 and confirmed RIN2’s function in recruiting BIN1 to early endosomes, a process that has been well-associated with AD etiology. Additionally, we performed quantitative interaction perturbation analyses on AD risk genes with AD-associated mutations or isoforms and identified significant changes in 91 protein interactions among 11 different protein variants. Finally, we found that subunits from the anaphase-promoting complex/cyclosome (APC/C), other novel BIN1 interactors identified by ADNeuronNet, mediated modulation of Tau-aggregation in neurons via regulation of APOE expression, uncovering a previously unrecognized BIN1-APC/C-APOE regulatory axis in AD pathobiology. Overall, these findings illustrate how our neuron-specific ADNeuronNet can be leveraged to uncover new risk gene candidates and cellular pathways that help advance our understanding of molecular mechanisms underlying AD risk.2026-07-312026-03-08PXD075357NEWT:6945NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:1211601NEWT:876138NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:3885NEWT:3888NEWT:1589NEWT:135622NEWT:6915NEWT:3649NEWT:101510NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:1000589NEWT:85963NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:286NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:294128NEWT:11676NEWT:55571NEWT:100226NEWT:4530NEWT:4896NEWT:75058NEWT:13616NEWT:1094343NEWT:296543NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:408172NEWT:408170NEWT:493760NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:460519NEWT:1187947NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:1182263NEWT:2711NEWT:376686NEWT:95486NEWT:9103NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:3635NEWT:5811NEWT:235443NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:32644NEWT:499175NEWT:109779NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:357NEWT:360094NEWT:470NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:7091NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:5180NEWT:256737NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1249668NEWT:7108NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:98334NEWT:426428NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:29204NEWT:2172103NEWT:507601NEWT:643680NCBITaxon:6157NEWT:746360NEWT:6239NEWT:470150NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NCBITaxon:2NEWT:985076NEWT:1215323NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:51329NEWT:243230NEWT:8355NEWT:9685NEWT:7029NEWT:1080772NEWT:1283300NEWT:6183NEWT:6063NEWT:334747NEWT:61235NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:260707NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NEWT:607699NEWT:6282NEWT:208964NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:5334NEWT:37334NEWT:382352NCBITaxon:10359NEWT:242619NEWT:632957NEWT:34073NEWT:373995NEWT:5689NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:36015NEWT:421932NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:61674NEWT:1117957NEWT:9913NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:180066NEWT:135588NEWT:1843183NEWT:58002NEWT:326423NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:1678078NEWT:749906NEWT:418985NEWT:146479NEWT:1911079NEWT:69373NEWT:264203NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:9612NEWT:38865NEWT:51953NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT:72407NEWT:349741NEWT:9605NEWT:9606NEWT:157295NEWT:641501NEWT:7668NEWT:915099NEWT:74940NEWT:9721NEWT:137221NEWT:1450511NEWT:1143193NEWT:411901NEWT:9838NEWT:105884NCBITaxon:9615NEWT:9839NEWT:58334NEWT:1193501NEWT:3055NEWT:58331NEWT:6326NEWT:6689NEWT:5476NEWT:299767NEWT:1274432NEWT:1274426NEWT:70448NEWT:9825NEWT:1274423NEWT:393765NEWT:698936NEWT:39946NEWT:9823NEWT:9940NEWT:521001NEWT:39947NEWT:1274420NEWT:578458