Changes in protein phosphorylation between post-embryonic seedlings and mature leaves.

<p>Phosphorylated proteins, peptides and residues (S = serine, T = threonine, Y = tyrosine) identified in post-embryonic seedlings and mature leaves of <i>Arabidopsis thaliana</i>. Common phosphopeptides with conserved phosphosites are highlighted in bold and common phosphopeptides with different phosphosites are highlighted in bold and italics.</p

Identification of Phosphoproteins in <i>Arabidopsis thaliana</i> Leaves Using Polyethylene Glycol Fractionation, Immobilized Metal-ion Affinity Chromatography, Two-Dimensional Gel Electrophoresis and Mass Spectrometry

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 <i>Arabidopsis</i> 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 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

List of identified phosphoproteins.

<p>Phosphorylated proteins, peptides and residues (phosphosites) identified by mass spectrometry in protein extracts from young <i>Arabidopsis</i> seedlings after rubisco depletion, IMAC enrichment and 2-DE separation of phosphoproteins. (a) <b>pS</b>, <b>pT</b> and <b>pY</b> = phosphorylated serine, threonine and tyrosine residues; M* = oxidized methionine. (b) Protein reported in the PhosPhAt 4.0 database. ^30,31 (c) Peptide reported in the PhoPhAt 4.0 database with a different protein phosphorylation site. ^{30 31} (d) Protein previously reported in <i>Arabidopsis</i> seedlings. ²³ (e) Peptide reported in the PhosPhAt 4.0 database with the same protein phosphorylation site. ^{30, 31} (f) Peptide previously reported in <i>Arabidopsis</i> seedlings with a different protein phosphorylation site. ²³ (g) Peptide previously reported in <i>Arabidopsis</i> seedlings with the same protein phosphorylation site. ²³</p><p>List of identified phosphoproteins.</p

Identification of phosphorylation sites using tandem mass spectrometry (MS/MS).

<p>The MS/MS spectra correspond to phosphopeptides with the following mass-to-charge (<i>m/z</i>) ratios, as obtained by trypsin digestion of proteins selected from the 2-DE gel shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130763#pone.0130763.g003" target="_blank">Fig 3</a> (A) <i>m/z</i> 689.810, showing phosphorylation of jacalin-lectin family protein (spot 32) at S195; (B) <i>m/z</i> 1134.612, showing phosphorylation of germin-like protein (spot 31) at T72; (C) <i>m/z</i> 814.872, showing phosphorylation of the Rubisco small subunit (spot 66) at T133; (D) <i>m/z</i> 936.502, showing phosphorylation of ribose 5-phosphate isomerase-related protein (spot 63) at S86. Peaks corresponding to sequential loss of intact amino acid residues from the C or N terminus of the peptide are labeled as b- or y-type ions, respectively.</p

Identification of Phosphoproteins in <i>Arabidopsis thaliana</i> Leaves Using Polyethylene Glycol Fractionation, Immobilized Metal-ion Affinity Chromatography, Two-Dimensional Gel Electrophoresis and Mass Spectrometry

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 <i>Arabidopsis</i> 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 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

Distribution and functional classification of identified phosphoproteins.

<p>(A) Numbers of phosphoproteins and phosphopeptides identified in post-embryonic <i>Arabidopsis</i> seedlings and mature <i>Arabidopsis</i> leaves,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130763#pone.0130763.ref011" target="_blank">11</a>] and of the phosphosites identified in phosphopeptides common to both tissues. (B) Functional classification of the phosphoproteins identified in <i>Arabidopsis</i> young seedlings according to the KEGG Pathway database (<a href="http://www.genome.jp/kegg/pathway.html" target="_blank">http://www.genome.jp/kegg/pathway.html</a>). Proteins involved in carbohydrate/energy metabolism, oxidative stress/redox regulation and photosynthesis/respiration account for over 50% of the identified phosphoproteome.</p

Plant phosphoproteome analysis using Rubisco depletion, IMAC enrichment of phosphoproteins, 2-DE and liquid chromatography-tandem mass spectrometry.

<p>Plant phosphoproteome analysis using Rubisco depletion, IMAC enrichment of phosphoproteins, 2-DE and liquid chromatography-tandem mass spectrometry.</p

Distribution of phosphorylated residues identified in plant proteins using immobilized metal-ion affinity chromatography of phosphorylated proteins or peptides.

<p>Distribution of phosphorylated residues identified in plant proteins using immobilized metal-ion affinity chromatography of phosphorylated proteins or peptides.</p

Identification of Phosphoproteins in <i>Arabidopsis thaliana</i> Leaves Using Polyethylene Glycol Fractionation, Immobilized Metal-ion Affinity Chromatography, Two-Dimensional Gel Electrophoresis and Mass Spectrometry

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 <i>Arabidopsis</i> 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 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

Two-dimensional gel electrophoresis of Rubisco-depleted phosphoproteins enriched by immobilized metal-ion affinity chromatography using PHOS-Select iron affinity gel beads.

<p>Phosphoproteins identified by liquid chromatography-tandem mass spectrometry are indicated using arrows and numbers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130763#pone.0130763.t001" target="_blank">Table 1</a>).</p