## 1 Introduction  
The completion of the genome sequencing in 2000 [1] has further propelled _Arabidopsis thaliana_ into one of the most well-established model organisms to study plant molecular biology/biochemistry [2]. _Arabidopsis_ is used for a wide range of \"OMICS\" analysis concerning genes (genomics; [3,4]), proteins (proteomics; [5, 6, 7]) and metabolites (metabolomics, [8]). One sub-topic of proteomics, rising in the last few years, is the field of phosphoproteomics [9]. The strong interest originates from the importance of protein phosphorylation for the biochemistry of all organisms, especially in regulating cellular processes, ranging from cell differentiation, development, cell cycle control, metabolism and signal transduction [10, 11, 12]. Probably 30% of all proteins are phosphorylated at any given time and state [13], indicating the immense dimension of the phosphoproteome. Beside its different roles in the regulation of protein synthesis, gene expression and apoptosis, phosphorylation events exhibit a pivotal role in defense responses [14]. An example is the activation of mitogen-activated protein kinase (MAPK)-mediated phosphorylation signaling cascades upon stress or other environmental signals [15, 16, 17]. The corresponding downstream targets of such a cascade are, to a great extent, unknown. For further understanding of defense mechanisms in plants, more knowledge about signaling cascades is of high significance. Therefore, a fully developed strategy for phosphoprotein/peptide enrichment is necessary.  
Unfortunately, plant phosphoproteomics using leaf material can be a challenging task. Not only the presence of highly abundant proteins, like RuBisCO, but also the low levels of phosphorylated signaling proteins limit their visualization and detection on PAGE-gels. Even highly advanced mass spectrometry is often unable to recover large numbers of phosphopeptides in complex samples. Common methods frequently describe the enrichment of phosphopeptides prior to measurement to overcome this challenge. Most methods use metal ions for the binding of phosphopeptides, for instance, chelated metal ions (immobilized metal affinity chromatography IMAC); [18, 19]) or metal oxides (metal oxide affinity chromatography (MOAC); [20]). Other methods describe the use of multi-step procedures, in which a first enrichment of phosphoproteins should assist the subsequent phosphopeptide enrichment [21]. Nevertheless, one disadvantage of such an approach is that not all phosphopeptides are efficiently captured, and also, information concerning the non-phosphorylated peptides is lost, which may impede target identification, for instance, in the cases of highly similar proteins of multigene families [22]. Other approaches first remove highly abundant proteins that might interfere with the applied phospho-enrichment matrix. In plants, this means the reduction or depletion of RuBisCO prior to phosphoprotein enrichment [23, 24]. A popular way to accomplish the fractionation of proteins is salting out with chemicals. Polyethylene-glycol (PEG)-based fractionation, for instance, has been successfully employed for improved proteome coverage, leading to the detection of differentially-expressed proteins of low abundance [25, 26]. However, since the remaining PEG can interfere in MS analysis, we tested here another commonly used fractionation, namely, ammonium sulfate (AS) precipitation. In previous work done in our laboratory, it could be shown that a reduction of the RuBisCO content via AS precipitation had a positive effect on the preparation of 2D-PAGE, as well as the enrichment of phosphoproteins [27]. As a further improvement for phosphoprotein analysis, we now incorporated the metal oxide affinity chromatography (MOAC) method [20] to the AS-based RuBisCO removal step, which, by itself, already acts as prefractionation/enrichment of phosphoproteins. This led to a facile, but efficient, phosphoproteome analysis procedure, which we termed prefractionation-assisted phosphoprotein enrichment (PAPE).