Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD061128/raw_files.rarftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD061128/spectronaut_output_files.rarftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD061128/spectronaut_search_file.rarftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD061128/experimental_design.rarprimaryOK200vince@wehi.edu.auMaria TanzerMass SpectrometryBottom-up proteomicsColon CancerDrugsE3 ligaseIntrabodyBafBiodegraderIntracellular antibodyXiapTnfIap proteinSwi/snfRingProtacSmac mimeticBioprotacCiap1Cancerhttps://www.ebi.ac.uk/pride/archive/projects/PXD061128Colorectal Adenocarcinoma Cell LineMalignant CellHT29 cell expressing different nanobody constructs were washed three times with ice-cold PBS, lysed in 2% sodium deoxycholate (SDC) (v/v) and 100 mM Tris-HCl (pH 8.5), and boiled immediately. After sonication, protein amounts were adjusted to 20 μg using the BCA protein assay kit. Samples were reduced with 10 mM (TCEP), alkylated with 40 mM 2-chloroacetamide, and digested with trypsin and lysC (1:50, enzyme/protein, w/w) overnight. Peptides were desalted using SDB-RPS-stage tips. Peptides were resolubilised in 5 μL 2% (v/v) ACN and 0.3% (v/v) TFA and 200 ng were injected into the mass spectrometer (TimsTOF Pro).PrideReconstituted peptides (1 µL) were separated by reverse-phase liquid chromatography on a 15 cm C18 fused silica column with an integrated emitter tip (IonOpticks, ID 75 µm, OD 360 µm, 1.6 µm C18 beads) using a custom nano-flow HPLC system (Thermo Ultimate 300 RSLC Nano-LC, PAL systems CTC autosampler). The HPLC was coupled to a timsTOF Pro (Bruker) equipped with a CaptiveSpray source. Peptides were loaded directly onto the column at a flow rate of 600 nL/min with 5% buffer A (99.9% Milli-Q water, 0.1% FA) and eluted at 400nL/min on a 30-min linear analytical gradient of increasing buffer B (90% ACN, 0.1% FA) from 2 to 34%, and 95% for 3 min at a flow rate of 400 nL/min. The timsTOF Pro (Bruker) was operated in diaPASEF mode using Compass Hystar 5.1. The settings on the TIMS analyzer were as follows: Lock Duty Cycle to 100% with equal accumulation and ramp times of 100 ms, and 1/K0 Start 0.6 V.·/cm2 End 1.6 V·s/cm2, Capillary Voltage 1400V, Dry Gas 3 l/min, Dry Temp 180°C. The DIA methods were set up using the instrument firmware (timsTOF control 2.0.18.0) for data-independent isolation of multiple precursor windows within a single TIMS scan. The method included two windows in each diaPASEF scan, with window placement overlapping the diagonal scan line for doubly and triply charged peptides in the m/z – ion mobility plane across 16 × 25 m/z precursor isolation windows (resulting in 32 windows) defined from m/z 400 to 1,200, with 1 Da overlap, and CID collision energy ramped stepwise from 20 eV at 0.85 V·s/cm2 to 59eV at 1.3 V·s/cm2. For proteome analysis, MS raw files were processed by the Spectronaut software version 19. Human uniport FASTA databases (42,351 entries, 2021) was used as forward databases. Cysteine carbamidomethylation was included as fixed modification and N-terminal acetylation and methionine oxidations were included as variable modifications. The false discovery rate (FDR) and PEP cutoff were set to less than 1% at the peptide and protein levels and a minimum length of seven amino acids for peptides was specified. Enzyme specificity was set as C-terminal to arginine and lysine as expected using trypsin and LysC as proteases and a maximum of two missed cleavages. Statistical tests were performed with Perseus. The 1D annotation-enrichment analysis detects whether expression values of proteins belonging to an enrichment term (here we used keywords, GOCC, GOMF, GOBP, and KEGG name) show a systematic enrichment or de-enrichment compared with the distribution of all expression values.ProteomicsJames VincePARTIALWalter and Elisa Hall Institute of Medical Research Inflammation DivisionHomo Sapiens (human)Not availabletanzer@wehi.edu.auWalter and Eliza Hall InstituteAustraliafalseIAP-binding intrabodies for concurrent degradation of proto-oncogenes and toggling cell death modalitiesSingle domain antibodies or antibody mimetics, such as monobodies, can be engineered to degrade undruggable targets via fusion to ubiquitin E3 ligases. In this study we developed multi-targeting biodegrader intrabodies by harnessing an inhibitor of apoptosis (IAP) E3 ligase RING domain, or the IAP-binding motif from SMAC/DIABLO. When fused with monobodies targeting the necroptotic death effector MLKL, IAP-binding intrabodies simultaneously targeted MLKL and IAPs for proteasomal degradation, thereby blocking necroptosis while sensitising cells to apoptosis. The potent degradative capacity of the IAP RING was demonstrated via its fusion to distinct antibody mimetics, with those targeting RAS enabling IAP and RAS proto-oncogenes to be concurrently eliminated. Quantitative proteomics identified that the anti-cancer potential of select IAP-binding intrabodies was due to their removal of the chromatin remodelling BAF complex. Consequently, we show that cancer cells exhibited IAP and BAF co-dependencies, with IAP RING intrabodies prolonging animal survival in an orthotopic breast cancer model. This work positions IAP-binding intrabodies as a novel class of biodegraders, with their multi-targeting capacity enabling the toggling of cell death modalities and eradication of cancer-drivers.2026-03-032025-02-24PXD061128NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:96731NEWT:383379NEWT:10029NEWT:913645NEWT:44491NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:3112NEWT:4442NEWT:192875NEWT:12637NEWT:59729NEWT:2164133NEWT:108061NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:5759NEWT:1736231NEWT:1392NEWT:498217NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:633149NEWT:10239NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:935293NEWT:375146NEWT:118698NEWT:263NEWT:118696NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:3702NEWT:243277NEWT:7067NEWT:118694NEWT:2850NEWT:118691NEWT:33548NEWT:274NEWT:408172NEWT:408170NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:1436733NEWT:460519NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:1513458NCBITaxon:40559NEWT:84588NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NCBITaxon:4932NEWT:644223NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:320637NEWT:983964NEWT:118499NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:352472NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:39491NCBITaxon:5811NEWT:29491NEWT:2014887NEWT:33952NEWT:261756NEWT:571256NEWT:40483NEWT:272634NEWT:9031NEWT:1872122NEWT:7091NEWT:141262NEWT:108931NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:7111NEWT:347515NEWT:9534NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:4093NEWT:4096NEWT:185579NEWT:941442NEWT:13076NEWT:7108NEWT:40479NEWT:4076NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:1268063NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:407821NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:28532NEWT:353152NEWT:8496NEWT:27448NEWT:1194669NEWT:243230NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:6289NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:469008NEWT:256318NEWT:9321NEWT:206411NCBITaxon:6191NEWT:596153NEWT:229533NEWT:1836NEWT:80863NEWT:105231NEWT:52638NEWT:57075NEWT:4097NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:1134506NEWT:38783NEWT:4006NEWT:6426NCBITaxon:5476NEWT:6669NEWT:9935NEWT:89453NEWT:287889NEWT:51515NEWT:588596NEWT:51511NEWT:8845NEWT:92867NEWT:92866NEWT:5334NEWT:51750NEWT:202950NEWT:382352NEWT:413071NEWT:632957NEWT:9925NEWT:89462NEWT:8839NEWT:90690NEWT:4232NEWT:2758385NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:312017NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:284593NEWT:100392NEWT:5315NEWT:9913NEWT:265872NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NEWT:999810NCBITaxon:5693NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:231490NEWT:430615NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1416333NEWT:1678078NEWT:749906NEWT:418985NEWT:1817808NEWT:749907NEWT:145263NEWT:694569NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:51953NEWT:3197NEWT:860688NEWT:13443NEWT:36329NEWT:62615NCBITaxon:3044782NEWT:160235NEWT:72407NEWT:349741NEWT:157295NEWT:2096NEWT:192222NEWT:9721NEWT:137221NEWT:9963NEWT:1617910NEWT:565050NEWT:2785785NEWT:411901NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:58331NEWT:6689NEWT:298315NEWT:70448NEWT:698936NEWT:9940NEWT:3168NEWT:521001NEWT:192011NEWT:1813887NEWT:118702NEWT:118700NEWT:224911NEWT:118705NCBITaxon:11552NEWT:6945NEWT:184922NEWT:6703NEWT:2104NEWT:2NEWT:157546NEWT:474186NEWT:38942NEWT:307972NEWT:1496NEWT:2102NEWT:45474NEWT:1590841NEWT:4513NEWT:1247NEWT:6938NCBITaxon:10407NEWT:1736309NEWT:4751NEWT:10360NEWT:47664NEWT:317548NEWT:15819NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:1239NEWT:36745NEWT:272563NEWT:82688NEWT:3885NEWT:3888NEWT:5821NEWT:44139NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:466585NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT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