Purification of MAP-kinase protein complexes and identification of candidate components by XL-TAP-MS.
Plant Physiol
; 187(4): 2381-2392, 2021 12 04.
Article
em En
| MEDLINE
| ID: mdl-34609515
ABSTRACT
The purification of low-abundance protein complexes and detection of in vivo protein-protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL-TAP-MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL-TAP-MS to study the MKK2-Mitogen-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-crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2-MPK4 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-protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL-TAP-MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein-protein interactions.
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Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
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Arabidopsis
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Quinases de Proteína Quinase Ativadas por Mitógeno
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Proteínas Quinases Ativadas por Mitógeno
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Proteínas de Arabidopsis
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Complexos Multiproteicos
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article