Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075809/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075809/PRC-7113DDA_UPRC_3702_H11119_2p_IonOpt18_25cm_Breusegem-shotgun-DDA-1_uncalibrated.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075809/PRC-7113DDA_UPRC_3702_H11119_2p_IonOpt18_25cm_Breusegem-shotgun-DDA-2_uncalibrated.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075809/PRC-7113DDA_UPRC_3702_H11119_2p_IonOpt18_25cm_Breusegem-shotgun-DDA-1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075809/PRC-7113DDA_UPRC_3702_H11119_2p_IonOpt18_25cm_Breusegem-shotgun-DDA-2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075809/out1dda.zipprimaryOK200frank.vanbreusegem@psb.vib-ugent.beJingjing HuangMass SpectrometryBottom-up proteomicshttps://www.ebi.ac.uk/pride/archive/projects/PXD075809RosetteTwo-week-old Arabidopsis seedlings from a stable overexpression line carrying the FRET(VRPR) biosensor under the CaMV 35S promoter and grown on half-strength MS agar were used for this assay. Each sample consisted of five seedlings. Sample 1 represents wounded tissue, generated by grinding the seedlings in liquid nitrogen and allowing the material to thaw for 5 minutes. Sample 2 consists of untreated seedlings and serves as the control. Proteins were extracted and resuspended in 5% SDS in 50 mM TEAB (pH 8), heated at 95 °C for 5 minutes, and sonicated for 30 minutes using a PIXUL® ultrasonicator (Active Motif). Proteins were then reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) and alkylated with 40 mM chloroacetamide (CAA). Digestion was performed with LysC endoproteinase using the suspension trapping (S-Trap) method (Protifi). After desalting, peptides were analyzed on an Ultimate 3000 ProFlow nanoLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a pneu-Nimbus dual ion source (Phoenix S&T). Peptides were separated using a non-linear gradient starting at 0.5% solvent B and reaching 26% solvent B (0.1% formic acid in acetonitrile) at 75 minutes, 44% at 95 minutes, and 56% at 100 minutes, followed by a 5-minute wash at 56% solvent B and re-equilibration with solvent A (0.1% formic acid in water). The flow rate was maintained at 250 nL/min. The mass spectrometer was operated in data-dependent acquisition (DDA) mode. Full MS scans (m/z 350–1200) were acquired in the Orbitrap at a resolution of 120,000. The top 12 precursor ions were isolated in the quadrupole (2 Da window), fragmented using higher-energy collisional dissociation (HCD), and analyzed in the Orbitrap at a resolution of 15,000, with the first mass set to m/z 120.PrideNot availableRaw LC–MS/MS data were processed using FragPipe v24 (https://github.com/Nesvilab/FragPipe). The full amino acid sequence of the biosensor construct, FRETGSlinker2, was manually appended to the Arabidopsis thaliana reference proteome database (https://www.uniprot.org/proteomes/UP000006548; 27,463 proteins) prior to analysis. Database searching was performed with MSFragger, using semi‑specific LysC digestion to enable identification of non‑LysC cleavage events. Iodoacetamide (IAM) alkylation of cysteine residues was specified as a fixed modification (carbamidomethylation, +57.02146 Da), while protein N‑terminal acetylation (+42.0106 Da) and methionine oxidation (+15.9949 Da) were included as variable modifications. Peptide length was constrained to 7–35 amino acids. Rescoring was carried out using MSBooster, and label‑free quantification was performed with IonQuant, including match‑between‑runs. Annotated MS/MS spectra were inspected using the integrated FragPipe‑PDV viewer for spectrum‑centric validation. All filtered PSM, peptide, and protein identification tables were exported in standard formats for downstream statistical analysis.Note: The FRETVRPR sensor appears as “FRETGSLinker2” in the FASTA file and downstream analysis outputs.ProteomicsFrank Van Breusegem1 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium 2 Center for Plant Systems Biology, VIB, 9052 Ghent, BelgiumArabidopsis Thaliana (mouse-ear Cress)PARTIAL10.1093/PLCELL/KOAG162jingjing.huang@ugent.beDepartment of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 71, 9052 Ghent, Belgium VIB Center for Plant Systems Biology, 9052 Ghent, BelgiumBelgiumfalseShedding light on plant proteolysis: genetically encoded fluorescent sensors as tools for profiling protease activitiesProteolysis is a universal process, as proteases play a pivotal role in modulating numerous signaling pathways. By hydrolyzing peptide bonds within their target proteins, proteases control their fate and function. Understanding the temporal and spatial dynamics of proteolytic events, including the proteases that execute them, is crucial for elucidating their particular roles across diverse biological processes. In this study, we developed and characterized a set of genetically encoded Förster resonance energy transfer (FRET)-based reporters for the detection of various proteolytic activities in plants. Our sensors reliably reported the activity of specific proteases, exhibiting performance comparable to established detection systems. In addition, we engineered variants capable of detecting the spatial dynamics of metacaspase-triggered proteolysis after wounding and during programmed cell death in roots. The implementation of these tools in plant research opens new opportunities to explore proteolytic mechanisms with enhanced precision. We demonstrate the feasibility of the FRET-based sensors to detect various activities in vivo with high spatiotemporal resolution. Overall, these biosensors constitute a versatile toolbox for probing protease function within its native cellular context, paving the way for deeper insights into plant biology and signaling. This dataset includes a targeted shotgun proteomics experiment designed to identify the specific endoproteolytic cleavage products generated from the FRET(VRPR) biosensor.2026-07-042026-03-18PXD075809NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2102NEWT:2042546NEWT:259447NEWT:45351NEWT:43179NEWT:4513NEWT:180454NEWT:5722NEWT:1247NEWT:1129NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:224326NCBITaxon: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:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4442NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:44586NEWT:626528NEWT:3347NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:1392NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:172NEWT:296543NEWT:316435NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:2850NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:3827NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:572307NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:54571NEWT:9598NEWT:8030NEWT:9483NCBITaxon:40559NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT: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:317NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:451516NEWT:63459NEWT:1276815NEWT:36185NEWT:588858NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:986NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:51329NEWT:40559NEWT:243230NEWT:8355NEWT:633NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:632NEWT:6183NEWT:6063NEWT:630NEWT:49240NEWT:334747NEWT:61235NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:1719NEWT:703612NEWT:260705NEWT: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