## 3 Results and Discussion
### Pape: Prefractionation-Assisted Phosphoprotein Enrichment  
Crude extract and the 40% AS fraction (40% AS) were subjected to phosphoprotein enrichment with metal oxide affinity chromatography (MOAC) [20] (Figure 1A) with minor modifications, as described in the Experimental section. To evaluate the reproducibility and efficiency of the PAPE procedure (a combination of AS precipitation followed by MOAC), the total extract, the 40% ASfraction and the corresponding MOAC-enriched fractions were each prepared three times, separated on a 1D-PAGE and visualized by coomassie brilliant blue and Pro-Q Diamond phosphospecific staining (Figure 1C). As observed in the stepwise fractionation, the non-fractionated samples had the least visible phosphoprotein content (crude extract). While a faint enrichment effect could be achieved via MOAC (crude extract + MOAC), the prefractionation (40% AS) already had a high phosphoprotein content, which was dramatically increased in combination with the additional MOAC phosphoprotein enrichment step (40% AS + MOAC, Figure 1C). We will hereafter refer to this \"40% AS + MOAC\" fraction as the PAPE fraction.  
Figure 1: Prefractionation-assisted phosphoprotein enrichment (PAPE). (**A**) Experimental setup. _Arabidopsis_ soluble leaf proteins were either extracted with phenol to obtain the crude extract (total protein) or pre-fractionated by 40% ammonium sulfate precipitation. Subsequently, phosphoproteins were enriched using metal oxide affinity chromatography (MOAC). Quality control was assessed with SDS-PAGE and phospho-specific Pro-Q Diamond staining. Mass spectrometry analysis was performed with on-line nano-LC (Easy-nLC II) FT-mass spectrometry (LTQ Orbitrap Velos); (**B**) SDS-PAGE showing the crude extract and stepwise ammonium sulfate (AS) fractionations. Each step was performed three times (lanes labeled 1, 2 and 3). Visualization of proteins was achieved with (I) Pro-Q Diamond phosphoprotein staining in false-color representation and (II) colloidal coomassieie staining. Protein molecular weights are indicated on the left-hand margin. (**C**) SDS-PAGE of different extraction and phosphoprotein enrichment steps, with visualization of the proteins as described above. Boxed areas mark the position of the large subunit of RuBisCO. (**D**) Boxplot depiction of protein yield (log10 microgram of proteins) from 25 g of leaf material (n = 6).  
Notably, on the basis of the prepared replicates shown here, the \"MOAC-only\" method (crude extract + MOAC) had a larger variability in phosphoprotein enrichment compared to the other procedures (Figure 1C, upper panel; see, also, the standard deviation of the box plot in Figure 1D). Moreover, the \"MOAC-only\" fractions contained substantial amounts of RuBisCO (Figure 1C, lower panel), which may be a hindrance in subsequent mass spectrometry-based detection of less abundant proteins [33]. The PAPE fraction showed no distinct bands, but a uniform distribution across all molecular masses in the coomassieie, as well as in the phosphospecific stain. Hence, the combination of 40% AS fractionation served both to remove RuBisCO and to enrich for phosphoproteins. The final protein yield by the PAPE procedure is about 0.6% of the total crude extract (Figure 1D); assuming all these are phosphoproteins, this is in agreement with the total phosphoprotein amount expected.