Updated project metadata. Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events. MS raw data were searched against an A. thaliana protein database based on The Arabidopsis Information Resource (TAIR) 10 with the Proteome Discoverer 1.3 using an in-house Mascot server (precursor mass tolerance: 7 ppm; fragment mass tolerance: 0.8 Da; missed cleavages: 2). Carbamidomethylation of cysteine was set as a static modification. Variable modifications were oxidation (Methionine), acetylation (protein N-terminus), deamidation (Asparagine/Glutamine) and phosphorylation (Serine/Threonine). Further data evaluation was carried out with the Scaffold software (Version 3.3, Proteome Software Inc., Portland, OR, USA), Proteome Discoverer 1.3 with phosphoRS 1.0 (Thermo Fisher Scientific) and DanteR [30] for total protein content. Phosphopeptides were identified with the Proteome Discoverer 1.3 software, which includes the phosphoRS 1.0 algorithm (Thermo Fisher Scientific) for phospho-site mapping. A false discovery rate (FDR) was calculated by searching a ―decoy‖ database containing all the target database sequences in reverse order. Peptide-spectrum match (PSM) was set at a q-value <0.05 (i.e., a corrected significance threshold employing the Benjamini-Hochberg FDR procedure to control for a family-wise error rate). Protein grouping was enabled.