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Samples were divided into two portions. One portion was irradiated at 365 nm ~15 cm away from a 365 nM LED lamp (UHP-T-365-MP, Prizmatix) in an 18-well glass-bottom plate (Ibidi) on an ice-cold metal block for 10 min and the other portion was preserved in the dark on ice. Samples were resolved by SDS-PAGE, 10% NuPAGE Bis-Tris polyacrylamide 1.5 mM gels used to load sample volumes above 20 µL. Proteins were visualized by Coomassie staining.   MS sample preparation by in-gel tryptic digestion.  Proteins were diluted in NuPAGE LDS Sample Buffer (Invitrogen) to 1.2x operating concentration, separated by SDS-PAGE and visualised using filtered Coomassie blue in a sterile dish. The protein bands of interest were cut out of the gel and into ~1mM cubes. The gel pieces were de-stained overnight in 50mM ammonium bicarbonate (ABC) and 50% Acetonitrile (ACN). Proteins were reduced with 10 mM DTT for 30 mins and alkylated in 50 mM iodoacetamide (VWR) in the dark for 30 mins. Gel pieces were washed with 100mM ABC and then 20 mM ABC, 50% ACN before being dehydrated with 100% ACN for 5 mins. Gel pieces were dried in a fume hood for 15 mins. Trypsin (Thermo) was added (1:100 w:w) according to estimated protein amounts in each band. Trypsin was diluted in 20 mM ABC, 9% ACN and added to the gel pieces in a volume that just covered the pieces (rehydration volume - RV). Gel pieces were incubated for 16 h at 37◦C. To extract the peptides, the rehydration volume (RV) of 100% ACN was added to the gel pieces, followed by RV 5% formic acid, 50% ACN. Pieces were dehydrated again by adding RV 100% ACN. The liquid was evaporated in the gyrovap using the “V-AQ” setting at 45°C. Peptide pellets were resuspended in 0.1% trifluoroacetic acid (TFA) 0.5% acetic acid and submitted for MS analysis. Gel pieces were shaken at RT, 1400 rpm during all steps unless stated otherwise.   LC-MS/MS peptide analysis Samples were analyzed on a Thermo Fisher Scientific Lumos Tribrid mass spectrometer coupled with a Thermo Dionex Ultimate 3000 RSLC HPLC. The buffers used for HPLC were 0.1% formic acid as buffer A and 80% acetonitrile (ACN) with 0.08% formic acid as buffer B. Trap column Acclaim pepmap 100 (C5, 100 μM × 2 cm) was used before the main column for sample concentration and cleanup. The peptide samples were loaded onto the trap column using a loading pump with 3% ACN and 0.1% TFA at a flow rate of 5 μl/min. The main column used was EASY- Spray column (C18, 2 μm, 75 μm × 50 cm) with a nano electrospray emitter built in. The flow rate of 300 nl/min was maintained throughout the run. Peptides were separated with a 90 min gradient as detailed within raw data files (see “Data availability” for further details). The separated peptides were analyzed on the mass spectrometer with the following settings. Spray voltage was 2 kV, RF lens level was 30%, and ion transfer tube temperature was 275°C. The mass spectrometer was operated in data-dependent mode with 2 s cycle time. The full scan was performed in the range of 375 to 1500 mass/charge ratio (m/z) at a nominal resolution of 120,000 at 200 m/z and automatic gain control (AGC) was set to 400,000 with a custom maximum injection time of 50 ms. This was followed by the selection of the most intense ions above an intensity threshold of 20000 for higher-energy collision dissociation (HCD) fragmentation, with normalized collision energy set to 30. MS2 scans were acquired for charge states 2 to 7 using an isolation width of 1.6 m/z. MS2 scans were done at resolution 60K using an AGC target of 500,000 and a maximum fill time of 500 ms. Dynamic exclusion was set to 30s."],"repository":["Pride"],"modification":[""],"quantification_method":[""],"data_protocol":["Identification of N-maleimido-diazirine-linked peptides   Data analysis was performed with MaxQuant version 2.4.0.0. Default settings were used with a few exceptions. A database of all the recombinant proteins included in the crosslinking assays was used. Digestion was set to Trypsin/P (ignoring lysines and arginine N-terminal to prolines) with a maximum of 8 missed cleavages. Match between runs was enabled. Prior to running the search, two new crosslinkers were added to MaxQuant; Hydrolysed N-maleimido-diazirine named ‘NMD169’: Linked composition H(11)O(3)C(8)N, mass 169.0738932246 Da. N-maleimido-diazirine named ‘NMD151’: Linked composition H(9)O(2)C(8)N, mass 151.0633285383 Da. Specificity 1 was C, position in peptide 1 was set to anywhere. Protein N-term 1 and C-term 1 were selected. Specificity 2 set for any amino acid (ACDEFGHIKLMNPQRSTVWY), position in peptide 2 was anywhere, protein N-term 2 and protein C-term 2 were selected. To search for crosslinked peptides the crosslinker NMD (non-cleavable) was selected, minimum length for a paired sequence was set to 3 and the maximum peptide mass 12,000 Da. The minimum peptide length for unspecified peptide search was 8 and maximum 25. The search included both intra-protein and inter-protein crosslinked peptides. Oxidation (M), Acetyl (Protein N-term) and Carbamidomethyl (C) were included as variable modifications, with a maximum of 5 per peptide allowed. First search was performed with Oxidation (M) and Acetyl (Protein N-term). Protein and crosslinked peptide level FDR was set to 1%."],"omics_type":["Proteomics"],"labhead":["Ronald T. Hay"],"instrument_platform":[""],"labhead_affiliation":["Professor of Molecular Biology. University of Dundee. UK"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"publication":["42112908 Chandler SF, Tatham MH, Branigan E, Nakasone MA, Makukhin N, Ciulli A, Hay RT. Photocross-linking activity-based probes to capture the dynamics of ubiquitin RING E3 ligase interactions. Biochem J. 2026 483(7):1115-1130 10.1042/bcj20260213"],"submitter_mail":["m.tatham@dundee.ac.uk"],"submitter_affiliation":["University of Dundee"],"submitter_country":["United Kingdom"],"pubmed_abstract":["Almost all cellular processes are influenced by ubiquitination. A large family of enzymes known as E3 ligases provides the specificity for ubiquitination, with the largest class among them, the Really Interesting New Gene (RING) E3s, comprising over 600 members in humans. RING E3s facilitate transfer of ubiquitin (Ub) to substrates by constraining the highly dynamic E2-Ub thioester linkage to be primed for attack from the substrate nucleophile. We have established a workflow that uses an N-maleimido diazirine (NMD) photoactivatable cross-linker attached to ubiquitin that, once stably linked to the active site of an E2, creates an activity-based probe (ABP) to monitor interactions with E3 ligases. Cross-linking mass spectrometry using the NMD-Ub-E2 ABP identified regions of interaction between ubiquitin and a selection of different RING E3s, which not only agreed with existing crystal structures, but was also used to evaluate in silico structural models of complexes yet to be resolved by conventional means. The cross-linking data also provided insight into domains of conformational flexibility which likely adopt multiple configurations in solution and which are challenging to monitor by other methods. NMD-Ub-E2 ABPs offer great potential to explore the ensemble conformations of E2-E3 complexes in solution and have scope for applications beyond the ubiquitin system."],"pubmed_title":["Photocross-linking activity-based probes to capture the dynamics of ubiquitin RING E3 ligase interactions."],"pubmed_authors":["Chandler Sarah F SF, Tatham Michael H MH, Branigan Emma E, Nakasone Mark A MA, Makukhin Nikolai N, Ciulli Alessio A, Hay Ronald T RT"],"additional_accession":[]},"is_claimable":false,"name":"Photocrosslinking Activity-Based Probes to Capture the Dynamics of Ubiquitin RING E3 Ligase Interactions","description":"Almost all cellular processes are influenced by ubiquitination. A large family of enzymes known as E3 ligases provide the specificity for ubiquitination, with the largest class among them, the RING E3s, comprising over 600 members in humans. RING E3s facilitate transfer of ubiquitin to substrates by constraining the highly dynamic E2-Ub thioester linkage to be primed for attack from the substrate nucleophile. We have established a workflow that uses a modified ubiquitin carrying a photoactivatable crosslinker that once stably linked to the active site of an E2, creates an activity based probe (ABP) to monitor interactions with E3 ligases. Using this, regions of interaction between ubiquitin and a selection of different RING E3 were determined, which not only confirmed existing structures of E2-Ub-RING-E3 complexes but was also used to assess new Ub-E2-E3 models generated in absence of existing structures.","dates":{"publication":"2026-06-08","submission":"2026-03-13"},"accession":"PXD075613","cross_references":{"TAXONOMY":["NEWT:1773","NEWT:3555","NEWT:38783","NEWT:1182590","NEWT:2","NEWT:10090","NEWT:935293","NEWT:749200","NEWT:35554","NEWT:4120","NEWT:5693","NEWT:347515","NEWT:1216979","NEWT:307972","NEWT:92867","NEWT:990346","NEWT:544496","NEWT:5334","NEWT:145953","NEWT:257309","NEWT:284812","NEWT:115104","NCBITaxon:1313","NEWT:43330","NEWT:67825","NEWT:44544","NEWT:13076","NEWT:373995","NEWT:544404","NEWT:3702","NEWT:8839","NEWT:4232","NEWT:990119","NEWT:1736309","NEWT:4113","NEWT:7227","NEWT:11298","NEWT:7469","NEWT:885318","NEWT:171101","NEWT:4081","NEWT:876138","NEWT:554","NEWT:5691","NEWT:408170","NEWT:260710","NEWT:106592","NEWT:237561","NEWT:9913","NEWT:10036","NEWT:4100","NEWT:7574","NEWT:1351","NEWT:1076","NEWT:6763","NEWT:7215","NEWT:803","NEWT:8030","NEWT:380394","NEWT:272563","NEWT:507601","NEWT:1639","NEWT:188229","NEWT:4909","NCBITaxon:79857","NEWT:746360","NEWT:6239","NEWT:1589","NEWT:135588","NEWT:135622","NEWT:216257","NEWT:6915","NEWT:9986","NEWT:101510","NEWT:95486","NEWT:3880","NEWT:58002","NEWT:9103","NEWT:4577","NEWT:5664","NEWT:2157","NEWT:146479","NEWT:1000589","NEWT:145943","NEWT:85962","NEWT:160488","NEWT:317447","NEWT:3635","NEWT:7955","NCBITaxon:2","NEWT:235443","NEWT:985076","NEWT:7959","NEWT:2261","NEWT:3197","NEWT:9615","NEWT:884019","NEWT:4565","NEWT:1264690","NEWT:169963","NCBITaxon:38727","NEWT:36329","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:9606","NEWT:367830","NEWT:243230","NEWT:931281","NEWT:373153","NEWT:7029","NEWT:1283300","NEWT:334747","NEWT:470","NCBITaxon:79824","NCBITaxon:4563","NEWT:3218","NEWT:5759","NEWT:9838","NCBITaxon:9615","NEWT:1736231","NEWT:1193501","NEWT:6287","NEWT:2242","NEWT:6326","NEWT:9796","NEWT:2762","NEWT:5476","NEWT:562","NEWT:260707","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1836","NEWT:1735272","NEWT:29760","NEWT:260705","NEWT:80863","NEWT:1148","NEWT:4932","NEWT:70448","NEWT:9825","NEWT:3603","NEWT:698936","NEWT:2759","NEWT:39946","NEWT:11676","NEWT:9823","NEWT:100226","NCBITaxon:6073","NEWT:4530","NEWT:4896","NEWT:6279","NEWT:7370","NEWT:573","NEWT:6282","NEWT:7091","NEWT:1134506"],"pubmed":["42112908"],"ORCID":["0000-0001-6986-1691"]}}