Publication Information
Mithoe et al., 2012
Abstract
J Proteome Res. 2012 Jan 1;11(1):438-48. doi: 10.1021/pr200893k. Epub 2011 Dec
1.
Targeted quantitative phosphoproteomics approach for the detection of
phospho-tyrosine signaling in plants.
Mithoe SC(1), Boersema PJ, Berke L, Snel B, Heck AJ, Menke FL.
Author information:
(1)Molecular Genetics, Department of Biology, Faculty of Science, Utrecht
University, Utrecht, The Netherlands.
Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal
systems. However, a few studies have also reported Tyr phosphorylation in
plants, but the relative contribution of tyrosine phosphorylation to plant
signal transduction has remained an open question. We present an approach to
selectively measure and quantify Tyr phosphorylation in plant cells, which can
also be applied to whole plants. We combined a (15)N stable isotope metabolic
labeling strategy with an immuno-affinity purification using phospho-tyrosine
(pY) specific antibodies. This single enrichment strategy was sufficient to
reproducibly identify and quantify pY containing peptides from total plant cell
extract in a single LC-MS/MS run. We succeeded in identifying 149 unique pY
peptides originating from 135 proteins, including a large set of different
protein kinases and several receptor-like kinases. We used flagellin perception
by Arabidopsis cells, a model system for pathogen triggered immune (PTI)
signaling, to test our approach. We reproducibly quantified 23 pY peptides in 2
inversely labeled biological replicates identifying 11 differentially
phosphorylated proteins. These include a set of 3 well-characterized flagellin
responsive MAP kinases and 4 novel MAP kinases. With this targeted approach, we
elucidate a new level of complexity in flagellin-induced MAP kinase activation.
DOI: 10.1021/pr200893k
PMID: 22074104 [Indexed for MEDLINE]
