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

Abstract

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.

Temperature-induced changes in the wheat phosphoproteome reveal temperature-regulated interconversion of phosphoforms

“…1C, D). Therefore, to identify early phosphorylation-controlled signalling components in wheat that are associated with a mild increased temperature, we subjected both leaf and spikelet samples from the 60 min time point to our phosphoproteomic workflow (Vu et al , 2016). …”

“…To our knowledge, this is currently the largest set of identified phosphosites in the Triticum family. The identified phosphosites in this study were added to the PTMViewer (bioinformatics.psb.ugent.be/webtools/ptm_viewer/) (Vu et al , 2016). In addition, we found several phosphosites that were differentially regulated between normal (21 °C or 24 °C) and increased ambient temperature (34 °C) in wheat leaves and spikelets (Fig.…”

Early mannitol-triggered changes in the Arabidopsis leaf (phospho)proteome reveal growth regulators

“…Subsequent proteome and phosphoproteome analyses were performed as previously described for protein extraction and trypsin digestion (Nikonorova et al , 2018 a ) and phosphopeptide enrichment (Vu et al , 2016) (see also Supplementary Protocol). For samples harvested 4 h after transfer to mannitol, protein extraction was performed by adding 100 µl of protein extraction buffer [5% ethylene glycol, 25 mM Tris–HCl pH 7.6, 15 mM MgCl 2 , 5 mM EGTA pH 8, 150 mM NaCl, 15 mM para-nitrophenylphosphate, 60 mM β-glycerophosphate, 1 mM DTT, 0.1% NP-40, 0.1 mM Na 3 VO 4 , 1 mM NaF, 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 µg ml −1 leupeptin, 10 µg ml −1 aprotinin, 10 µg ml −1 soybean trypsin inhibitor, 0.1 mM benzamidine, 5 µg ml −1 antipain, 5 µg ml −1 pepstatin, 5 µg ml −1 chymostatin, 1 µM E64] and successive rounds of vortexing and freezing in liquid nitrogen.…”

“…A 50 µl aliquot of the peptide mixtures was used for LC-MS/MS analysis. LC-MS/MS analysis was performed as previously described (Vu et al , 2016). Proteome and phosphoproteome samples were analysed using 3 h gradients on a quadrupole Orbitrap instrument (Q Exactive).…”

Transcriptional integration of paternal and maternal factors in the Arabidopsis zygote

“…The leaf samples were harvested, and the phospho-enrichment experiment was performed as described previously (Vu et al 2016). Detailed methods are in the Supplemental Material.…”