## 3 Results and Discussion
### Validation of Phosphoprotein Enrichment by the PAPE Procedure  
Figure 1C demonstrated that the PAPE procedure precipitated and enriched phosphoproteins. To further support this Pro-Q Diamond phosphostain evidence (Figure 1C), we determined if there was indeed an increase in the identification of known phosphoproteins from the fractionation steps. Using P3 DB, a curated plant phosphoprotein database that contains only experimentally verified high quality entries, we found that the 40% AS, MOAC and PAPE fractions contain significantly higher numbers of known phosphoproteins than the crude extract (Figure 3B). Since the number of identified proteins varied between fractions, we also calculated the identified known phosphoproteins as a percentage of all identified proteins within each fraction (grey line in Figure 3B) in order to circumvent any misrepresentation. This demonstrated that the PAPE fraction had proportionally more known phosphoproteins than the MOAC fraction (36% and 23%, respectively), thus suggesting the improvement of the PAPE procedure over MOAC alone to enrich phosphoproteins. Gene ontology (GO) annotation of the proteins identified in the PAPE fraction showed an enrichment of proteins involved in response to abiotic and biotic stimuli and to stress (Figure 3A). Since protein phosphorylation regulates many of these processes, it supports the effectiveness of PAPE to enrich lowly abundant phosphorylated proteins that are also involved in cellular signaling. Correspondingly, we identified more phosphopeptides in the 40% AS, MOAC and PAPE fractions than in the crude extract (Figure 3C). In particular, when represented as the percentage of  
Figure 3: Changes of the protein/phosphoprotein composition. (**A**) Gene ontology functional categorization (based on the The Arabidopsis Information Resource (TAIR) gene ontology (GO) web-tool) of the proteins detected with the PAPE procedure; (**B**) The number of identified proteins in the various fractionation steps that are annotated as known phosphoproteins in the P3 DB database. The grey line represents the percentage of identified known phosphoproteins to the total number of identified proteins in each fraction (see Figure 2A); (**C**) The number of phosphopeptides identified in the various fractionation steps. (Only high-confidence phosphopeptides with a phosphorylation site probability (pRS) score \(>\)30 are considered; for a full list, see Table S2). Each experiment was performed three times and measured twice. Black bars are the average number of phosphopeptides (\(+\)/–standard deviation) detected in each fraction, while grey bars depict the total number of non-identical phosphopeptides identified from all replicates. The grey line depicts the percentage of identified phosphopeptides to the total number of identified proteins in each fraction. phosphopeptides relative to the total number of proteins identified in each fraction, more phosphopeptides were recorded in the PAPE than the MOAC fraction (6.2% and 4.5%, respectively). Interestingly, many of the phosphopeptides detected in the PAPE fractions were not listed in the P3 DB [36,37] and PhosPhAt 3.0 [38,39] databases, which includes both novel phosphopeptides in proteins that are, so far, not annotated as phosphoproteins, as well as novel phosphopeptides in other regions of known phosphoproteins (see Tables 1 and S2). Note that Table 1 lists only the novel phosphopeptide with a high-confidence pRS score cutoff (>30); a longer list of all potential phosphopeptides is shown in Table S2. Additionally, Table S3 (a modified version of Table S2) links the identified phosphopeptides and the associated phosphoproteins. Inspection of these tables also reveals a progressive increase in the number of phosphopeptides associated with a particular (phospho)protein from the crude extract to the final PAPE fraction.