Enrichment and Analysis of Intact Phosphoproteins in Arabidopsis Seedlings

Abstract

Protein 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 only 18 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.

Fig 1. Plant phosphoproteome analysis using Rubisco depletion, IMAC enrichment of phosphoproteins, 2-DE and liquid chromatography-tandem mass spectrometry.

Fig 2. One-dimensional gel electrophoresis of the flow-through and bound protein fractions (10 μg) obtained following depletion of plant protein extracts using Seppro Rubisco IgY spin columns.

Molecular weight markers (M) are shown on the left.

Fig 3. Two-dimensional gel electrophoresis of Rubisco-depleted phosphoproteins enriched by immobilized metal-ion affinity chromatography using PHOS-Select iron affinity gel beads.

Phosphoproteins identified by liquid chromatography-tandem mass spectrometry are indicated using arrows and numbers (see Table 1).

Fig 4. Distribution and functional classification of identified phosphoproteins.

(A) Numbers of phosphoproteins and phosphopeptides identified in post-embryonic Arabidopsis seedlings and mature Arabidopsis leaves,[11] and of the phosphosites identified in phosphopeptides common to both tissues. (B) Functional classification of the phosphoproteins identified in Arabidopsis young seedlings according to the KEGG Pathway database (http://www.genome.jp/kegg/pathway.html). Proteins involved in carbohydrate/energy metabolism, oxidative stress/redox regulation and photosynthesis/respiration account for over 50% of the identified phosphoproteome.

Fig 5. Identification of phosphorylation sites using tandem mass spectrometry (MS/MS).

The MS/MS spectra correspond to phosphopeptides with the following mass-to-charge (m/z) ratios, as obtained by trypsin digestion of proteins selected from the 2-DE gel shown in Fig 3 (A) m/z 689.810, showing phosphorylation of jacalin-lectin family protein (spot 32) at S195; (B) m/z 1134.612, showing phosphorylation of germin-like protein (spot 31) at T72; (C) m/z 814.872, showing phosphorylation of the Rubisco small subunit (spot 66) at T133; (D) m/z 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.