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HothornData-dependent acquisitionMass Spectrometryhttps://www.ebi.ac.uk/pride/archive/projects/PXD073452Protein samples were loaded on a mini polyacrylamide gel, migrated and stained by Coomassie Blue. Gel bands were excised and digested with sequencing-grade trypsin (Promega, Madison, WI, USA, Product N. V5111) or chymotrypsin (Promega, Prod.N. V1061) essentially as described (Shevchenko, et al., 2006). Extracted tryptic peptides were dried and resuspended in 0.1% formic acid, 2% (v/v) acetonitrile. LC-MS/MS protocol for first part “Quantitative phosphoproteomics of protein kinases”: LC-MS/MS analyses were carried out on a TIMS-TOF Pro (Bruker, Bremen, Germany) mass spectrometer interfaced through a nanospray ion source (“captive spray”) to an Ultimate 3000 RSLCnano HPLC system (Dionex). Peptides were separated on a reversed-phase custom packed 45 cm C18 column (75 μm ID, 100Å, Reprosil Pur 1.9 um particles, Dr. Maisch, Germany) at a flow rate of 250 nl/min with a 2-27% acetonitrile gradient in 33 min followed by a ramp to 45% in 5 min and to 90% in 5 min (total method time: 65 min, all solvents contained 0.1% formic acid). Data-dependent acquisition was carried out using a standard TIMS PASEF method (Meier, et al., 2018) with ion accumulation for 100 ms for each the survey MS1 scan and the TIMS-coupled MS2 scans. Duty cycle was kept at 100%. Up to 10 precursors were targeted per TIMS scan. Precursor isolation was done with a 2 or 3 m/z windows below or above m/z 800, respectively. The minimum threshold intensity for precursor selection was 2500. If the inclusion list allowed it, precursors were targeted more than one time to reach a minimum target total intensity of 20’000. Collision energy was ramped linearly based uniquely on the 1/k0 values from 20 (at 1/k0=0.6) to 59 eV (at 1/k0=1.6). Total duration of a scan cycle including one survey and 10 MS2 TIMS scans was 1.16 s. Precursors could be targeted again in subsequent cycles if their signal increased by a factor 4.0 or more. After selection in one cycle, precursors were excluded from further selection for 60s. Mass resolution in all MS measurements was approximately 35’000. LC-MS/MS protocol for second part “Identification of phosphosites in kinases and phosphatases”: Data-dependent LC-MS/MS analyses of samples were carried out on a Fusion Tribrid Orbitrap mass spectrometer (Thermo Fisher Scientific) interfaced through a nano-electrospray ion source to an Ultimate 3000 RSLCnano HPLC system (Dionex). Peptides were separated on a reversed-phase custom packed 45 cm C18 column (75 μm ID, 100Å, Reprosil Pur 1.9 um particles, Dr. Maisch, Germany) with a 4-90% acetonitrile gradient in 0.1% formic acid at a flow rate of 250 nl/min (total time 140 min). Full MS survey scans were performed at 120'000 resolution. A data-dependent acquisition method controlled by Xcalibur software (Thermo Fisher Scientific) was used that optimized the number of precursors selected (“top speed”) of charge 2+ to 5+ while maintaining a fixed scan cycle of 0.6 s. Peptides were fragmented by higher energy collision dissociation (HCD) with a normalized energy of 32%. The precursor isolation window used was 1.6 Th, and the MS2 scans were done in the ion trap. The m/z of fragmented precursors was then dynamically excluded from selection during 60 s.PrideData processing protocol for first part “Quantitative phosphoproteomics of protein kinases”: Scaffold (version Scaffold 5.0.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 80.0% probability by the Percolator posterior error probability calculation (Käll, et al. 2008, Bioinformatics 24(16): i42-8). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, et al. 2003, Anal. Chem. 75 (17): 4646-58). All phosphopeptide identifications and phosphosite localizations reported were manually validated. Data processing protocol for second part “Identification of phosphosites in kinases and phosphatases”: MS data were analyzed using Mascot 2.8 (Matrix Science, London, UK) set up to search the Arabidopsis_thaliana reference proteome based on the UniProt database (www.uniprot.org, version of August 24th, 2020, containing 39’347 sequences), and a custom contaminant database containing the most usual environmental contaminants and enzymes used for digestion (keratins, trypsin, etc, 351 sequences). In a second pass search, the sequence of the expressed tagged proteins was searched alone, together with the contaminant database, and the matched peptides were used for peptide quantitation. Trypsin (cleavage C-terminal to K,R) , allowing 2 missed cleavages and Chymotrypsin (cleavage C-terminal to Phe, Leu, Trp, Tyr, semispecific) were used as the enzyme definitions. Mascot was searched with a parent ion tolerance of 15 ppm and a fragment ion mass tolerance of 0.5 Da. Carbamidomethylation of cysteine was specified in Mascot as a fixed modification. Protein N-terminal acetylation, methionine oxidation and phosphorylation on Ser, Thr and Tyr were specified as variable modifications. Based on Mascot results, the MsViz software (Martin-Campos, et al. 2017, J. Proteome Res 16(8): 3092–3101) was used to manually quantify and compare intensities of phosphorylation sites in the BSU1 sequence. Reference unmodified peptides were used for normalizing protein amounts when necessary.ProteomicsMichael HothornStructural Plant Biology Laboratory, Department of Plant Sciences, University of Geneva, 1211 Geneva, SwitzerlandCOMPLETEArabidopsis Thaliana (mouse-ear Cress)42166246 Rico-Resendiz F, Pri-Tal O, Raia P, Moretti A, Chen H, Yu J, Broger L, Fuchs C, Hothorn LA, Loubéry S, Hothorn M. Plant Kelch phosphatases are Ser/Thr phosphatases involved in cell cycle regulation. Proc Natl Acad Sci U S A. 2026 123(21):e2600591123 10.1073/pnas.2600591123michael.hothorn@unige.chDepartment of Plant Science, University of GenevaSwitzerland10.6019/PXD073452falseIdentification of phosphorylations sites in Arabidopsis thaliana BSU1 recombinantly expressed in insect cells, and when incubated with the GSK3 kinase BIN2.Brassinosteroids (BRs) are plant steroid hormones sensed at the cell surface by the membrane receptor kinase BRI1. Activation of BRI1 leads to the dephosphorylation of BZR1/BES1 transcription factors through inactivation of the glycogen synthase kinase 3 BIN2. Here, the in vitro phosphorylation sites of the isolated BSU1 Kelch domain (residues 11-351), phosphatase domain (residues 421-793) and of full-length BSU1 (residues 1-793) recombinantly expressed in baculovirus-infected Spodoptera frugiperda cells were identified in isolation (labelled ctrl) or after incubation with the GSK3 kinase BIN2, expressed as a thioredoxin A fusion protein in E. coli. Reactions were performed in the presence of 100 micromolar ATP and 1 mM MgCl2 for 1:30 h at room temperature.2026-05-222026-01-23PXD073452NEWT:6945NEWT:184922NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:1211601NEWT:876138NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:3885NEWT:3888NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NEWT:1390363NEWT:288705NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:374990NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:286NEWT:391619NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:294128NEWT:11676NEWT:55571NEWT:100226NEWT:4530NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:296543NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:105023NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:408172NEWT:408170NEWT:493760NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:460519NEWT:1187947NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:1182263NEWT:2711NEWT:376686NEWT:95486NEWT:9103NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:3635NEWT:5811NEWT:235443NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:32644NEWT:225117NEWT:499175NEWT:109779NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:357NEWT:360094NEWT:470NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:7091NEWT:108931NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1249668NEWT:7108NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:98334NEWT:426428NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:29204NEWT:2172103NEWT:507601NEWT:643680NCBITaxon:6157NEWT:746360NEWT:6239NEWT:470150NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:243230NEWT:8355NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:6183NEWT:6063NEWT:630NEWT:334747NEWT:61235NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NEWT:4097NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NEWT:607699NEWT:6282NEWT:208964NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:5334NEWT:37334NEWT:382352NCBITaxon:10359NEWT:242619NEWT:632957NEWT:34073NEWT:373995NEWT:5689NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:37349NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:36015NEWT:421932NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:61674NEWT:1117957NEWT:9913NEWT:8709NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:180066NEWT:817NEWT:135588NEWT:1843183NEWT:58002NEWT:326423NEWT:36111NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:1678078NEWT:749906NEWT:418985NEWT:146479NEWT:749907NEWT:1911079NEWT:69373NEWT:264203NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:9612NEWT:38865NEWT:51953NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:112273NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT:1440772NEWT:72407NEWT:1355477NEWT:349741NEWT:9605NEWT:9606NEWT:90371NEWT:157295NEWT:641501NEWT:7668NEWT:915099NEWT:7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