Up-to-Date Workflow for Plant (Phospho)proteomics Identifies Differential Drought-Responsive Phosphorylation Events in Maize Leaves

Protein
phosphorylation is one of the most common post-translational
modifications (PTMs), which can regulate protein activity and localization
as well as protein–protein interactions in numerous cellular
processes. Phosphopeptide enrichment techniques enable plant researchers
to acquire insight into phosphorylation-controlled signaling networks
in various plant species. Most phosphoproteome analyses of plant samples
still involve stable isotope labeling, peptide fractionation, and
demand a lot of mass spectrometry (MS) time. Here, we present a simple
workflow to probe, map, and catalogue plant phosphoproteomes, requiring
relatively low amounts of starting material, no labeling, no fractionation,
and no excessive analysis time. Following optimization of the different
experimental steps on Arabidopsis thaliana samples,
we transferred our workflow to maize, a major monocot crop, to study
signaling upon drought stress. In addition, we included normalization
to protein abundance to identify true phosphorylation changes. Overall,
we identified a set of new phosphosites in both Arabidopsis
thaliana and maize, some of which are differentially phosphorylated
upon drought. All data are available via ProteomeXchange with identifier
PXD003634, but to provide easy access to our model plant and crop
data sets, we created an online database, Plant PTM Viewer (bioinformatics.psb.ugent.be/webtools/ptm_viewer/), where all phosphosites identified in our study can be consulted.