Enrichment and analysis of intact phosphoproteins in arabidopsis seedlings

  1. Get@NRC: Enrichment and analysis of intact phosphoproteins in arabidopsis seedlings (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1371/journal.pone.0130763
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Journal titlePLoS ONE
Article numbere0130763
Subjectphosphoprotein; ribulosebisphosphate carboxylase; threonine; tyrosine; Arabidopsis thaliana; carbohydrate metabolism; immobilized metal affinity chromatography; liquid chromatography; oxidative stress; photosynthesis; plant development; protein phosphorylation; proteomics; seedling; tandem mass spectrometry; two dimensional gel electrophoresis
AbstractProtein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P3DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.
Publication date
AffiliationNational Research Council Canada; NRC Plant Biotechnology Institute; Aquatic and Crop Resource Development
Peer reviewedYes
NPARC number21276976
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Record identifiera4dd6ad7-79d4-4382-83df-37b26c393c40
Record created2015-11-10
Record modified2016-05-09
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