## 3 Results and Discussion
### Validation of Phosphoprotein Enrichment by the PAPE Procedure
\begin{table}
\begin{tabular}{r l l l l l l l l l l} \hline \hline \multicolumn{1}{c}{**No.**} & \multicolumn{1}{c}{**Protein code**} & \multicolumn{1}{c}{**Description**} & \multicolumn{1}{c}{**Sequence**} & \multicolumn{1}{c}{**MH+[Da]**} & \multicolumn{1}{c}{**q-Value**} & \multicolumn{1}{c}{**PEP**} & \multicolumn{1}{c}{**pRS Score**} & \multicolumn{1}{c}{**\# PSMs**} & \multicolumn{1}{c}{**pRS Site Probabilities**} \\ \hline
9 & AT1G74920.1; & aldehyde dehydrogenase & & & & & & & & & \\ AT5G48170.1 & 10A89 & SPLIVFDVDDLK & 1,555.73259 & 0.009502 & 0.2584146 & 70 & 2 & S(1): 100.0 \\
10 & AT3G28710.1 & ATPase, V0/A0 complex, & & & & & & & & \\  & & subunit C/D & & & & & & & & \\  & & GDSL-like & & & & & & & & \\
11 & AT1G73610.1 & Lipase/Aeyhydrolase & SYSTEMAPQIENKAK@n & 1,977.93410 & 0.017056 & 0.3870838 & 30 & 18 & S(1): 50.3; V(2): 50.3; T(4): 99.3 & \\  & & superfamily protein & & & & & & & & \\
12 & AT2G41110.1 & calmodulin 2 & ADQLTDDQSEFK & 1,589.66288 & 0.019757 & 0.513722 & 50 & 5 & T(5): 100.0; S(10): 0.0 \\  & & RNA-binding & & & & & & & & \\
13 & AT1G70200.1 & (REMRBD/RNP motifs) & QFTGQSLAFGKVKQIK & 1,973.05405 & 0.02041 & 0.4607051 & 40 & 7 & T(3): 50.0; S(6): 50.0 \\  & & family protein & & & & & & & \\  & & S-adenosyl-I-methionine- & & & & & & & \\
14 & AT2G22400.1 & dependent & EIRKNQTER & 1,366.68921 & 0.02499 & 0.5231273 & 38 & 1 & T(7): 100.0 \\  & & methyltransferases & & & & & & & & \\  & superfamily protein & & & & & & & & \\
15 & AT4G30630.1 & unknown protein & & & & & & & & \\  & & & & & & & & & \\
16 & AT5G64090.1 & unknown protein & & & & & & & & \\  & & P-loop containing & & & & & & & & \\  & & nucleoside triphosphate & & & & & & & & \\
17 & AT1G80380.3 & & & & & & & & & \\  & & hydrolases superfamily & & & & & & & & \\  & protein & & & & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._ \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline
**No.** & **Protein code** & **Description** & **Sequence** & **MI+[Da]** & **q-Value** & **PEP** & **pBS Score** & **\# PSMs** & **pBS Site Probabilities** \\ \hline
6 & AT3G18780.2 & actin 2 & & & AEDDIQPIVCDNGTGMVKAG & & & & & & \\  & & & & & & & & & & \\
7 & AT5G09810.1; & actin 1/7 & & & & & & & & & \\ AT2G37620.1 & & & & & & & & & & & \\ \hline
8 & AT5G56180.1 & actin-related protein 8 & & & & & & & & & \\  & & & & & & & & & & \\  & & & & & & & & & & \\  & & & & & & & & & & \\  & & & & & & & & & & & \\
9 & AT1G49240.1 & actin 8 & & & & & & & & & & \\  & & & & & & & & & & \\  & & & & & & & & & & & \\  & & & & & & & & & & & \\
10 & AT3G02830.1 & zinc finger protein 1 & & & & & & & & & & \\  & & & & & & & & & & \\
11 & AT1G64790.1 & ILITYHA & SPIVSAAFAFNLVK & 1,525,75934 & 0.017784 & 0.3293382 & 48 & 5 & S(1): 10.3, S(5): 89.7 & & & \\
12 & AT4G38770.1 & proline-rich protein 4 & & & & & & & & & \\  & & & & & & & & & & \\
13 & AT4G31120.2 & SHKI binding protein 1 & DVHLGIFPTTATPNMSW** & 2,095,92093 & 0.031227 & 0.430492 & 31 & 1 & T(12): 14, S(17): 97.6 & & \\  & & & & & & & & & & \\  & & & & & & & & & & \\
14 & AT5G16330.1 & protein-related & & & & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._However, there are also cases where no phosphopeptides could be identified for the putative phosphoprotein enriched in the PAPE fraction (e.g., MPK4 or MPK6). This is possibly one of the caveats of the present study, which is that when compared to the reproducibility in identification of the (putative) phosphoprotein (see Table S1, Figure 2B), there is often difficulties or variation in the phosphopeptide identification between replicate measurements. Contrary to expectation, the absolute number of phosphopeptide identified is not particularly high, despite the increased phosphoprotein detection (Figure 3C). However, such limitations can be attributed to the fact that the subsequent tryptic digestion reintroduced a complex peptide mixture, thereby hindering the phosphopeptide identification by MS as a consequence of the over-representation of non-phosphorylated peptides over phosphopeptides [40].