Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report-first-pass.gg_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report.unique_genes_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report-first-pass.stats.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report.stats.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report.gg_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report-first-pass.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report-first-pass.pr_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report.pg_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report.pr_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report-first-pass.pg_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report-first-pass.unique_genes_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report.log.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/report.pdfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag15m_GG_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag60m_GG_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag15m_GG_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag30m_GG_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag60m_GG_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag15m_GG_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dmso_GG_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag30m_GG_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag60m_GG_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dtag30m_GG_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dmso_GG_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/PP2CA_dmso_GG_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag60m_GG_R3_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag30m_GG_R3_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag30m_GG_R2_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag60m_GG_R1_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag15m_GG_R1_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dmso_GG_R2_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag15m_GG_R3_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag30m_GG_R1_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dmso_GG_R1_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag60m_GG_R2_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dtag15m_GG_R2_raw.quantftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD061232/C__Gajanan_time_dTAG-PP2CA_GG_TP_GG_PP2CA_dmso_GG_R3_raw.quantprimary200OKg.sapkota@dundee.ac.ukGajanan SatheMass SpectrometryData-independent acquisitionUbiquitinomeDtagTpdhttps://www.ebi.ac.uk/pride/archive/projects/PXD061232dTAG/dTAGPPP2CA knock-in HEK293 cells generated using CRISPR/Cas9 genome editing. The cells were treated with dTAG13 for 0,15,30 and 60 min. The cells were lysed by 8M urea buffer in 50 mM TEABC, both buffers contain protease and phosphatase inhibitor cocktail tablets. All steps for urea lysis were carried out at room temperature to avoid carbamylation. Proteins were measured by BCA protein estimation method. The described amount of proteins were used for in solution digestion. For urea lysis protein were reduced using 5mM DTT for 60 min at RT, followed by alkylation using 10 mM CAA for 20 min at RT in dark. This were incubated with LysC for 4h in (1:100) enzyme to protein ratio. After LysC the lysate were diluted with 50 mM TEABC to reduce the concentration of urea less than 2M. Trypsin with (1:20) enzyme to substrate were used for the in-solution digestion and incubated 12-16 hr at RT. The reaction was quenched with the addition of trifluoroacetic acid (TFA) to give a final concentration of 1% TFA (v/v) and samples were desalted on 200 mg Sep-Pak C18 cartridges (Waters). For Sep-Pak clean-up, the following solvents were prepared fresh: activation solvent (100% (v/v) acetonitrile (ACN)); Solvent-1 (0.1% (v/v) TFA); Solvent-2 (0.1% (v/v) formic acid (FA)); Solvent-3 (50% (v/v) ACN in 0.1% (v/v) FA). Sep-Pak cartridges were equilibrated with 5 mL 100% ACN, followed by 5 mL 50% ACN, 0.1% FA and finally with 5 mL 0.1% TFA twice. Samples were then loaded onto the equilibrated C18 cartridges, washed with 5 mL 0.1% TFA four times, followed by washing with 5 mL 0.1% FA. Samples were then eluted with 6 mL 50% ACN, 0.1% FA. Desalted samples were then dried to completeness in a SpeedVac concentrator. These dried peptides were used for the DiGly immunoprecipitation. For agarose bead enrichment, cross-linked antibodies were used. The antibodies were washed with IAP buffer. The dried peptides were dissolved in 1 mL of IAP buffer. The peptide solution was then centrifuged at 12,000 rpm for 10 minutes, and the supernatant was added to agarose beads cross-linked with DiGly antibodies. These solutions were incubated on a rotator at 4°C for 2 hours with end-over-end rotation. After incubation, the beads were washed twice with 1 mL of IP buffer, followed by an additional wash with cold Milli-Q® water. After removing all the supernatant, the beads were incubated with 200 µL of 0.15% TFA at room temperature while shaking at 1,400 rpm. After a brief spin, the supernatant was recovered and desalted using in-house prepared, 200 µL two-plug StageTips. These peptides were analyzed on an Orbitrap Exploris 480 mass spectrometer interfaced with Dionex Ultimate 3000 nanoflow liquid chromatography system. The dried peptides were reconstituted in 0.1% formic acid and loaded on a trap column (Acclaim PepMap100 C18, 100 μm × 2 cm, 5 μm, 100 Å) and separated on an analytical column (Acclaim PepMap RSLC C18, 75 μm × 50 cm, 2 μm, 100 Å) at a flow rate of 250 nL/min using a step gradient of 0–8% solvent B (90% ACN/0.1% FA) for the first 12 min, followed by 8–25% up to 110 min, 25-37% for 110-125 min. The total run time was set to 145 min. For DIA MS, scan resolution was set at 120,000, scan range was set between 375-1500, and normalised AGC target was set at 300%. For MS2 the ions were fragmented using HCD with stepped energy of 10 and 25, AGC target set at 3000%, and variable windows were used for MS2 acquisition.PrideNot availableThe raw data files acquired in DIA mode were processed using DIA-NN, version 1.8 (https://github.com/vdemichev/DiaNN). Human uniport was used as protein sequence database. Two missed cleavage and a maximum of two variable modifications per peptide were allowed (acetylation of protein N-termini and oxidation of methionine). Carbamidomethylation of cysteines was set as fixed modification and K-GG was added. This data analysis was carried out using library-free analysis mode in DIA-NN by enabling “deep learning-based spectra and RTs prediction”. Match between the runs (MBR ) was enabled .ProteomicsProf Gopal SapkotaMRC PPU University of DundeePARTIALHomo Sapiens (human)Not availablegsathe001@dundee.ac.ukCentre for Targeted Protein Degradation (CeTPD) University of DundeeUnited KingdomfalseData-independent acquisition approach for comprehensive ubiquitinome profiling in targeted protein degradationTargeted protein degradation (TPD) has emerged as a highly promising therapeutic strategy for a wide range of diseases, including cancer and neurodegenerative disorders. The ubiquitin-proteasome system, which is responsible for protein degradation, plays a critical role in this process. Gaining comprehensive insights into the landscape of cellular ubiquitylation events is essential for the development of selective and efficient targeted protein degradation approaches. In recent years, data-independent acquisition (DIA) approach has gained significant popularity as a robust and unbiased approach for quantitative proteomics. By leveraging DIA, we have developed a cutting-edge workflow that utilizes diGly antibody-based enrichment followed by an optimized Orbitrap-based DIA method for the identification of ubiquitylated peptides. Through the implementation of our workflow, we have identified over 40,000 diGly precursors corresponding to more than 7,000 proteins in a single measurement from cells exposed to a proteasome inhibitor, highlighting an exceptional throughput. By applying our optimized workflow, we successfully identify ubiquitylation sites on substrate proteins in various TPD approaches, thereby helping establish mode of action for TPD. Our workflow therefore holds tremendous potential for rapidly establishing mode of action for various TPD modalities, such as for PROTACs and molecular glues.2026-06-092025-02-26PXD061232NEWT: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:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT: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: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:243277NEWT:990119NEWT:2850NEWT: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:9483NEWT:1513458NCBITaxon:40559NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:508771NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:29158NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:1715989NEWT:476272NEWT:3747NEWT:195051NEWT: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:1276815NEWT:36185NEWT:588858NEWT:1225786NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:197221NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:89764NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:2059687NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:986NEWT:52641NEWT:7038NEWT:7159NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:119466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