Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/ChBL_116.pr_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_5_TEX_R2_Slot1-47_1_3840.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_6_TEX_R3_Slot1-48_1_3841.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_7_PEX_R1_Slot1-49_1_3842.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_8_PEX_R2_Slot1-50_1_3843.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/TEFF_R1_Slot1-43_1_3906.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_9_PEX_R3_Slot1-51_1_3844.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_12_TRM_R3_Slot1-54_1_3847.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/TEFF_R2_Slot1-45_1_3907.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_4_TEX_R1_Slot1-46_1_3835.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_10_TRM_R1_Slot1-52_1_3845.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063865/116_11_TRM_R2_Slot1-53_1_3846.d.zipprimaryOK200e1bennett@ucsd.eduXuezhen GeMass SpectrometryBottom-up proteomicsData-independent acquisitionProteostasisImmunotherapyTumorCd8+ t cellE3 ubiquitin ligaseTumor-infiltrating lymphocyteCancerhttps://www.ebi.ac.uk/pride/archive/projects/PXD063865T CellCytotoxic T-lymphocyteCell pellets, each containing 105 sorted cells, were resuspended in 50 μL of freshly prepared urea lysis buffer (8 M urea, 75 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM β-glycerophosphate). The suspension was sonicated for 300 s (30 s on/30 s off cycles) in a water bath sonicator. Protein was reduced with 10 mM TCEP for 30 min, alkylated with 15 mM freshly prepared iodoacetamide (IAA) for 45 min in the dark, and then treated with 10 mM DTT for 15 min to quench excess IAA. The samples were diluted to a final volume of 400 μL with 50 mM Tris-HCl, pH 8.0. For digestion, 200 ng Lys-C and 400 ng sequencing grade modified trypsin were added to the samples, which were then incubated at 37 °C with shaking overnight. The digestion was terminated by adding formic acid to achieve a final concentration of 1%.The digests were desalted using the Stage-Tip method. Subsequently, 2 μL of the 10 μL peptide sample was analyzed using an LC-MS/MS setup comprising a timsTOF Pro 2 mass spectrometer (Bruker Daltonics) coupled to a nanoElute 2 nano-LC system (Bruker Daltonics) via a CaptiveSpray ion source. Peptides were first loaded onto a trap column (PepMap Neo, 5 mm x 300 μm Trap) and then eluted through a reversed-phase C18 column (PepSep, 25 cm x 150 μm, 1.5 μm) maintained at 50 °C over a 50 min gradient of 5-35% solvent B (acetonitrile in 0.1% formic acid) at a flow rate of 450 nL/min. The peptides were analyzed using data-independent acquisition (dia) Parallel Accumulation-Serial Fragmentation (PASEF) mode. The isolation windows for dia-PASEF were determined based on data-dependent acquisition (dda)-PASEF data92 and generated by implementing the py_diAID algorithm to maximize proteome coverage94. The settings for dda-PASEF included the following: a ramp time and an accumulation time of 75 ms each, one MS scan, and 10 PASEF MS/MS ramps per acquisition cycle. The MS survey scan covered 100-1700 m/z and 0.6-1.6 V⋅s/cm². Precursors with up to 5 charges were selected, with an active exclusion time of 0.4 min. For dia-PASEF, 50 variable isolation windows for 25 dia-PASEF scan cycles (two windows per cycle) were designed to encompass the precursor distribution across the m/z-1/K0 plane as defined by the dda-PASEF data, ranging from 323.6 to 1221.6 m/z and 0.7 to 1.34 V⋅s/cm², resulting in a cycle time of 2.11 s. Collision energy was decreased linearly with increasing mobility, starting from 59 eV at 1/K0 = 1.6 V⋅s/cm² and reducing to 20 eV at 1/K0 = 0.6 V⋅s/cm².PrideThe dia-PASEF raw files were processed using a library-free search approach in DIA-NN version 1.8.196. An in silico digestion of a UniProt Mus musculus reference proteome (downloaded in January 2023) was employed to predict the spectral library. The search parameters applied included the following: tryptic digestion with up to two missed cleavages, carbamidomethylation of cysteine as a static modification, and oxidation of methionine and N-terminal acetylation as variable modifications. The precursor false discovery rate (FDR) threshold was set at 1%. For quantification, the quantification strategy was set to Robust LC (high precision) and cross-run normalization was set to RT-dependent. Match-between-runs (MBR) was enabled. The resulting protein group matrix from DIA-NN, reported at a 1% FDR (global q-values for protein groups; global and run-specific q-values for precursors), was used for downstream and statistical analysis. The statistical analysis was performed using the limma package in R97.ProteomicsEric J BennettDepartment of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.PARTIALHomo Sapiens (human)x1ge@ucsd.eduNot availableUniversity of California San DiegoUnited StatesfalseProteostasis sustains T cell differentiation potential and tumor-infiltrating lymphocyte functionTumors that contain tumor-infiltrating lymphocytes (TIL) with tissue-resident memory T cells (TRM) features are associated with better patient prognoses. While CD8+ T cells that target tumors often fail to restrain tumor growth due to progressive T cell exhaustion, pathogen-specific TRM survey tissues and sustain protective capacity for years. We identified protein homeostasis (proteostasis)-related E3 ubiquitin ligase genes shared by TRM and progenitor-exhausted TIL (TPEX), that are lost as TIL become terminally-exhausted (TEX). Sustained ligase expression led to increased TCF1+ T cell accumulation and improved anti-tumor function and less T cell exhaustion in chronic infection. Loss-of-function in cancer models impaired TIL accumulation and function and altered T cell differentiation in response to acute infection. Low-input mass spectrometry revealed abundant expression of protein degradation pathways by TEX, yet they accumulated unfolded proteins. Ligase overexpression rescued unfolded protein accumulation in TEX, demonstrating their functional contribution to proteostasis. Ligase overexpression in combination with immune checkpoint blockade led to improved survival from murine melanoma, demonstrating that the relationship between proteostasis and T cell differentiation opens up new avenues for cancer immunotherapy.2026-04-212025-05-13PXD063865NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:71647NEWT:2NEWT:157546NEWT:474186NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:1496NEWT:544496NEWT:2102NEWT:2042546NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:4513NEWT:180454NEWT:5722NEWT:1247NEWT:6938NEWT:1129NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:44139NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4442NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NEWT:2164133NCBITaxon:183674NEWT:1316931NEWT:224308NEWT:167387NEWT:84645NEWT:44586NEWT:626528NEWT:3347NEWT:511145NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:431241NEWT:931281NEWT:4550NEWT:1000561NEWT:2133NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NEWT:145481NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:1392NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:246196NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:161934NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:5507NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:644042NEWT:296543NEWT:316435NEWT:42157NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:1034304NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:9378NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:1077286NEWT:943129NEWT:243277NEWT:990119NEWT:7067NEWT:2850NEWT:1111708NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:3827NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:930945NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:572307NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:1194599NEWT:272844NEWT:54571NEWT:9598NEWT:8030NEWT:1513458NCBITaxon:40559NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:537457NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:508771NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:29158NEWT:644223NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:1679NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NCBITaxon:138NEWT:40483NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:1428NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:1055524NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:187878NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:40479NEWT:317NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:273068NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:451516NEWT:63459NEWT:36185NEWT:588858NEWT:1225786NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:197221NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:434271NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:89764NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:38018NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:2059687NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:986NEWT:623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