Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry

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DOIResolve DOI: http://doi.org/10.1021/pr200917t
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TypeArticle
Journal titleJournal of Proteome Research
Volume11
Issue1
Pages425437; # of pages: 13
Subjectprotein IMAC; phosphorylation; polyethylene glycol; two-dimensional gel electrophoresis; Rubisco; photosystem
AbstractReversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods due to the relatively low abundance of these proteins, and in plants this is further complicated by the high abundance of Rubisco protein in green tissues. We present a novel method for plant phosphoproteome analysis that firstly depletes Rubisco by polyethylene glycol fractionation, and then utilizes immobilized metal-ion affinity chromatography to enrich for phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO2 fixation, protein assembly and folding, stress response, redox regulation and cellular metabolism. Both the large and small subunits of Rubisco were found to be phosphorylated at multiple sites, while the depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discoveries of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal phosphoproteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of signal transduction during growth and development in plants.
Publication date
LanguageEnglish
AffiliationNRC Plant Biotechnology Institute; National Research Council Canada
Peer reviewedYes
NRC number50188
NPARC number19115365
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Record identifier038210eb-f0cb-4310-b862-682f91992037
Record created2012-04-17
Record modified2016-05-09
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