We apply a gel-free phosphoproteomics pipeline to different biological systems: wheat and soybean organs, Arabidopsis cell suspension cultures, and Arabidopsis seedlings. We combine these systems with loss- and gain-of-function approaches (such as tightly controlled systems using a constitutively active form under a native, inducible promoter), engineered kinases, and specific stimuli to perform an untargeted mass spectrometric analysis of the phosphoproteome. To confirm the importance of key differentially phosphorylated proteins (ideally hubs controlling major switches in physiological and developmental processes) in a biological process, candidates are functionally characterized, e.g. using kinase assays, to demonstrate functionality and in vivo phosphorylation, respectively, complemented with loss- and gain-of-function approaches (including site-directed phospho-site mutagenesis) and detailed analyses of expression patterns, both in Arabidopsis and monocot crops when relevant.
In addition to using this for our own research questions, we also offer our phosphoproteomics pipeline as a service. If you are interested, do not hesitate to reach out.
Key Publications:
Targeted Profiling of Protein Phosphorylation in Plants.
Xu X, Gevaert K, De Smet I, Vu LD.
Methods Mol Biol. 2023;2718:167-179.
Proteome Analysis of Arabidopsis Roots.
Nikonorova N, Vu LD, Stes E, Gevaert K, De Smet I.
Methods Mol Biol. 2018;1761:263-274.
Vu LD, Stes E, Van Bel M, Nelissen H, Maddelein D, Inzé D, Coppens F, Martens L, Gevaert K, De Smet I.
J Proteome Res. 2016 Dec 2;15(12):4304-4317.