We apply a gel-free phosphoproteomics pipeline to different biological systems: Arabidopsis cell suspension cultures, liquid Arabidopsis seedling cultures and the monocot leaves.
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 (Blethrow et al. 2008; Hengeveld et al. 2012), and specific stimuli to perform an untargeted mass spectrometric analysis of the phosphoproteome (e.g. Kline et al. 2010). 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 using kinase assays and phospho-specific antibodies 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.
- Hengeveld, R. C., Hertz, N. T., Vromans, M. J., Zhang, C., Burlingame, A. L., Shokat, K. M., & Lens, S. M. (2012). Development of a chemical genetic approach for human Aurora B kinase identifies novel substrates of the chromosomal passenger complex. Mol. Cell. Proteomics 11, 47-59.
- Kline, K. G., Barrett-Wilt, G. A., & Sussman, M. R. (2010). In planta changes in protein phosphorylation induced by the plant hormone abscisic acid. Proc. Natl. Acad. Sci. USA 107, 15986-15991.
- Blethrow, J. D., Glavy, J. S., Morgan, D. O., & Shokat, K. M. (2008). Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates. Proc. Natl. Acad. Sci. USA 105, 1442-1447.