Published: November 17, 2011 r2011 American Chemical Society 425 dx.doi.org/10.1021/pr200917t | J. Proteome Res. 2012, 11, 425–437 ARTICLE pubs.acs.org/jpr Identification of Phosphoproteins in Arabidopsis thaliana Leaves Using Polyethylene Glycol Fractionation, Immobilized Metal-ion Affinity Chromatography, Two-Dimensional Gel Electrophoresis and Mass Spectrometry Uma K. Aryal,* ,† Joan E. Krochko, and Andrew R. S. Ross ‡ Plant Biotechnology Institute, National Research Council, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9 b S Supporting Information 1. INTRODUCTION Recent improvements in protein analysis techniques, includ- ing mass spectrometry (MS), have brought proteomics to the forefront of methods for biological research. 1 Characterization of post-translational modifications of proteins is important for interpreting their biological functions and functional states. 2 Phosphorylation is one of the most important protein modifica- tions, affecting approximately one-third of the proteome and playing an important role in many cellular processes. 3 Although the study of protein phosphorylation holds particular promise for dissecting signaling pathways, the identification of phosphory- lated proteins and phosphorylation sites can be challenging because cellular levels of such proteins are often too low for detection using conventional methods. 4,5 The size, charge and hydrophobicity of tryptic phosphopeptides also make it difficult to identify and determine the stoichiometry of phosphorylation for individual proteins using MS. 4 Two-dimensional polyacrylamide gel electrophoresis (2D- PAGE) has the ability to resolve complex protein mixtures on the basis of two independent variables (isoelectric point and molecular mass) and has long been recognized as a key technology in proteome research. 6 It is particularly well-suited to detecting changes in protein phosphorylation since each phosphorylation event produces an incremental reduction in pI, resulting in characteristic horizontal rows of protein spots on 2D gels. 5 The combination of 2D-PAGE and MS provides a powerful platform for visualizing relative protein abundance and identifying proteins and their post-translational modifications in complex samples. Good sample preparation is the key to achieving reproducible, high quality gels 7 and recent improvements in sample prepara- tion, as well as protein visualization and MS techniques, 8 have led to a significant increase in the number of plant proteomic studies employing 2D-PAGE. 9 The plant proteome is highly dynamic, and more complex than the genome due to factors such as alternative splicing and posttranslational modification. 7 Plants also produce a large number of nonproteinaceous compounds that can interfere with 2D-PAGE separations. 10 These interfer- ing metabolites, which are particularly abundant in leaves and Special Issue: Microbial and Plant Proteomics Received: September 9, 2011 ABSTRACT: Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phospho- proteins 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. Sub- sequent protein separation by one- and two-dimensional gel electro- phoresis 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. KEYWORDS: protein IMAC, phosphorylation, polyethylene glycol, two-dimensional gel electrophoresis, Rubisco, photosystem