## 3 Results and Discussion
### Reproducibility and Robustness of PAPE on the Basis of Mass Spectrometry Analysis  
In addition to the coomassieie and phosphostain gel-based analysis, mass spectrometry may provide a more qualitative estimation of the PAPE efficiency. Tryp peptides derived from two micrograms of proteins from each of the three replicates of the four fractionation steps (_i.e._, crude extract, 40% AS fraction and the corresponding MOAC-treated samples of these two fractions) were measured with shotgun LC-MS. Each sample was measured in two LC-MS runs and the proteins identified for each fractionation step pooled from both runs. This led to the identification of 850, 1,024, 1,151 and 803 proteins from the crude extract, the 40% AS fraction and their corresponding MOAC-treated samples, respectively (SCAFFOLD Software parameters: minimum protein probability 99.0%/minimum number of peptides 2/minimum peptide probability 90%). These represent a total of 1,928 unique  
Figure 1: _Cont._  
proteins, and the distribution in the four fractionation steps is illustrated in Figure 2A. The identities of these 1,928 proteins are listed in Supplemental Table S1. Figure 2: Mass spectrometry analysis of proteins from the fractionation steps of the PAPE procedure. (**A**) Flower plot showing the qualitative differences in the protein composition of the various PAPE fractions. The numbers are the total number of proteins identified from three experiments, with each sample being measured twice; (**B**) Variability and reproducibility of the PAPE procedure. Each small square represents a scatter plot of protein abundance (quantitative values based on spectral counting, SCAFFOLD; DanteR [30]) of the intersecting samples from the various fractionation steps. The letters A-D denote crude extract, crude extract + MOAC, 40% AS and 40% AS + MOAC, respectively, and the numbers 1-3 correspond to the three replicate experiments for each fractionation step. Note the strong positive correlation within the three replicate experiments of each fractionation step (colored boxes); (**C**) Principal Component Analysis (PCA) plot. The dashed lines divide the plot into sectors along the weight of the principal components separating with/without prefractionation and MOAC phosphoprotein enrichment steps, respectively. The qualitative protein composition varied greatly between fractions. In fact, of the total 1,928 proteins identified, only 227 proteins were common to all fractions, thus suggesting that the fractions contain different subsets of proteins (Figure 2A). The overlap between the crude extract and the 40% AS fraction was 440 proteins (\(\sim\)50% of the crude extract), indicating that 40% AS precipitated a subset of the total proteins, as is expected when considering the wide range of protein solubility in aqueous solvents [34]. Surprisingly, the overlap between the crude extract and the MOAC-enriched fraction revealed 662 proteins, which represents \(\sim\)78% of the crude extract. Since it is unlikely that 78% of the identified proteins in the crude extract are phosphoproteins, it hints at substantial unspecific binding to the metal oxide. For instance, this might be due to binding to the negative charges provided by carboxylate moieties within proteins [35], which can exacerbate the binding of phosphoproteins in complex protein mixtures. These problems of the MOAC step in capturing non-phosphorylated targets is partially alleviated by the PAPE procedure described here, since the AS-prefractionation is already enriched for phosphoproteins (see Figure 1C). Therefore, the PAPE procedure is clearly advantageous compared to using only MOAC in phosphoproteomics. The high technical reproducibility of each fractionation step can be seen in the positive linear relationship in the scatter plot of the quantitative value (based on spectral counting, SCAFFOLD; DanteR [30]) of each identified protein between the replicate experiments (see the colored boxes in Figure 2B).