{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T10:41:45Z","timestamp":1790246505052,"version":"4.1.0"},"reference-count":42,"publisher":"Oxford University Press (OUP)","issue":"4","license":[{"start":{"date-parts":[[2021,9,21]],"date-time":"2021-09-21T00:00:00Z","timestamp":1632182400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"funder":[{"DOI":"10.13039\/501100001659","name":"German Research Foundation","doi-asserted-by":"crossref","award":["BE4054\/2-1"],"award-info":[{"award-number":["BE4054\/2-1"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01GM074830"],"award-info":[{"award-number":["R01GM074830"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,12,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>The purification of low-abundance protein complexes and detection of in vivo protein\u2013protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL\u2013TAP\u2013MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL\u2013TAP\u2013MS to study the MKK2\u2013Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde\u2013crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2\u2013MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein\u2013protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL\u2013TAP\u2013MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein\u2013protein interactions.<\/jats:p>","DOI":"10.1093\/plphys\/kiab446","type":"journal-article","created":{"date-parts":[[2021,9,17]],"date-time":"2021-09-17T11:31:00Z","timestamp":1631878260000},"page":"2381-2392","source":"Crossref","is-referenced-by-count":9,"title":["Purification of MAP\u2013kinase protein complexes and identification of candidate components by XL\u2013TAP\u2013MS"],"prefix":"10.1093","volume":"187","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9746-7162","authenticated-orcid":false,"given":"Franz","family":"Leissing","sequence":"first","affiliation":[{"name":"Department of Plant Physiology, RWTH Aachen University, Aachen 52056, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicola V","family":"Misch","sequence":"additional","affiliation":[{"name":"Department of Plant Physiology, RWTH Aachen University, Aachen 52056, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaorong","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Physiology & Biophysics, University of California, Irvine, California 92697, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linda","family":"Werner","sequence":"additional","affiliation":[{"name":"Department of Plant Physiology, RWTH Aachen University, Aachen 52056, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Physiology & Biophysics, University of California, Irvine, California 92697, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8503-1943","authenticated-orcid":false,"given":"Uwe","family":"Conrath","sequence":"additional","affiliation":[{"name":"Department of Plant Physiology, RWTH Aachen University, Aachen 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