test this, L cells were transfected with Pdx1 and the lysates were subsequently incubated with or without Lambda phosphatase and subjected to NIA analysis. The NIA profile from non-treated and phosphatase treated lysates were superimposed and revealed that the phosphatase treatment shifted the 6.0 peak to 6.1 while the 6.3 and 6.4 peaks remained unaffected (Fig. 4A). This suggests that Pdx1 harbors one primary phosphorylation represented by the 6.0 peak. The smaller peaks at 5.9 are also affected by the phosphatase treatment and it is likely that they represent additional but less abundant phosphorylations. To validate that the phosphatase treatment per se did not affect the NIA profile we analyzed the same lysates for the endogenous protein Hsp70 (Fig. 4B) and found the Hsp70 profiles for treated verses non-treated to be identical. Similar results were observed in bTC (Fig. 4C) and mouse islets (Fig. 4D) which express endogenous Pdx1. Serine 61 is the Primary Site of Phosphorylation in Pdx1 To test if the NIA assay could be used to map the phosphorylated residue in Pdx1 we carried out an alanine scan where all serines, tyrosines and threonines which are putative phosphorylation sites were replaced by alanine. Plasmids encoding the mutated Pdx1 proteins were transfected into L cells and aTC cells. The lysates were analyzed using NIA or western blots to confirm the expression and SDS-PAGE mobility of Pdx1 (Fig. 5A). To estimate the amount of phosphorylated Pdx1 we took advantage of the observation that the signal intensity of the 6.4 peak is unaffected by phosphorylation while the intensity of the 6.0 Post-Translational Modification of Pdx1 peak is related to the amount of phosphorylated protein. The ratio between the 6.4 and the 6.0 peak should therefore reflect the relative proportion of phosphorylated Pdx1. We find that only Pdx1S61A differs markedly from wild type Pdx1 (Fig. 5B) and superimposing the profile of Pdx1S61A onto the wild type profile revealed a marked reduction of the 6.0 peak (Fig. 5C). However, a residual 6.0 peak which could be removed by phosphatase treatment could still be detected, indicating the presence of at least one additional phosphorylation (Fig. 5D). To examine how constitutive phosphorylation affects the NIA profile of Pdx1 we replaced serine 61 with the negatively charged residue glutamic acid (Pdx1S61E), hereby mimicking a phosphorylation. When Pdx1S61E was analyzed using the NIA assay we only observed a single peak at pI 6.1(Fig. 5E) suggesting that the modifications of Pdx1 are absent if the protein is phosphorylated. This might also explain why the 6.3 and 6.4 peaks are unaffected following de- phosphorylation. Mass Spectrometry Analysis of Pdx1 The NIA analysis of Pdx1 identified S61 as the primary phosphorylation of Pdx1 in L cells, aTC cells and HEK293 cells (data not shown). To confirm this finding we purified Pdx1 protein from over-expressing HEK293 cells or from Min6 cells which express endogenous Pdx1. The purified protein was initially digested with trypsin. However, despite a 98% coverage of the Pdx1 protein, we were not able to conclusively detect the S61 phosphorylation. In order to investigate this discrepancy in Figure 4. Pdx1 is phosphorylated. In order to determine the identity of the peaks found in the Pdx1 profile we treated the lysate with lambda phosphatase to see if the removal of phosphorylations would shift the peaks. A-D) NIA profile (in 8 M urea) of the dephosphorylated lysate (red) is show superimposed on the control treated lysate (grey). A) Over expression of pdx1WT in L results in a shift of the 6.0 peak to 6.1, which fits the expected change in pI caused by a phosphorylation. The 6.40 peak is unaffected by the dephosphorylation. B) The NIA profile of Hsp70 from the same lysates serves as a control to show that the dephosphorylation assay does not impact the profile of a non phosphorylated protein. Control treatment or dephosphorylation of bTC cells (C) and mouse islets (D), show similar results. Results are representative of at least three independent experiments. doi:10.1371/journal.pone.0035233.g004 PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35233