title: Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry.
abstract: Reversible 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 is further complicated in plants by the high abundance of Rubisco in green tissues. We present a novel method for plant phosphoproteome analysis that depletes Rubisco using polyethylene glycol fractionation and utilizes immobilized metal-ion affinity chromatography to enrich 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 CO(2) fixation, protein assembly and folding, stress response, redox regulation, and cellular metabolism. Both large and small subunits of Rubisco were phosphorylated at multiple sites, and depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discovery of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal proteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of growth and development in plants.
authors.affiliation: Plant Biotechnology Institute, National Research Council, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9. uma.aryal@pnnl.gov
authors.forename: Uma K
authors.initials: UK
authors.lastname: Aryal
authors.forename: Joan E
authors.initials: JE
authors.lastname: Krochko
authors.forename: Andrew R S
authors.initials: AR
authors.lastname: Ross
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry*; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D029681:Arabidopsis Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D011092:Polyethylene Glycols; D012273:Ribulose-Bisphosphate Carboxylase