Post-Translational Modification of Pdx1 Figure 5. NIA profiles of Pdx1 alanine scanning shows Pdx1 to primarily be phosphorylated on serine 61. To identify putative phosphorylation sites in Pdx1, which may not have been picked up by the MS, we carried out an alanine scan. A) All serines, tyrosines and threonines in Pdx1 were mutated into alanines and transfected into L cells and aTC cells which are negative for endogenous Pdx1 expression and subjected to NIA analysis. B) Western blots against Pdx1 and b-Actin were used to confirm the expression of Pdx1 from the plasmids. Since the 6.4 peak is unaffected by dephosphorylation we used the area under curve (pI 6.4)/area under curve (pI 6.0) ratio to identify mutants where the phosphorylation was reduced. In most cases the intensity of the two bands are similar resulting in a relative ratio around 1 in both L cells (light grey bars) and aTC cells (dark grey bars). However, in Pdx1S61A the phosphorylated band (pI 6.0) was reduced and as a result the relative ratio increased to around 4. The screen was performed twice in each cell line and a representative result is shown. C-D) NIA profiles of S61 mutants (red) superimposed on the wild type Pdx1 profile (grey) (C). Also, dephosphorylation of the S61A mutant (red) superimposed on the control-treated lysate (grey), shows a residual peak at 6.0 which is removed by phosphatase (D).Mutating S61 to the phospho-mimic glutamic acid (E) clearly reduces the 6.0 peak. Note, that the entire Pdx1 profile of this mutant including the 6.3 and 6.4 peaks are moved to the left, as would be expected from the calculated pI change of an alanine to glutamic acid substitution. The NIA analysis in C-E were done in both L and aTC (data not shown) cells in least three independently transfected cell lysates, yielding similar results. doi:10.1371/journal.pone.0035233.g005 more detail we analyzed Pdx1 under chymotryptic treatment prior to mass spectrometric analysis. Using chymotrypsin ensures that Pdx1 is proteolytically cleaved at different amino acids (Phe, Ile, Tyr and Trp) as compared to when using trypsin (Lys and Arg). This allows for increased sequence coverage of the analyzed protein and hence increased analytical coverage of PTMs. Using this strategy we were able to significantly confirm the presence of S61 phosphorylation in both HEK293 cells (Fig. 6A) and in Min6 cells (Fig. 6B). To evaluate the occupancy rate for the identified phosphorylation sites we compared the peptide abundance of unmodified peptide to its phosphorylated counterpart (Fig 6C). Although this is not the most accurate measure it still provides information as to how extensive a given amino acid is phosphorylated. The comparison reveals that S61 indeed is the most highly abundant phosphorylation form in comparison to S269, with a ,12% S61 occupancy rate in HEK293 and a corresponding S61 occupancy rate of ,5% in Min6 cells. In comparison the occupancy rates for S269 phosphorylation in both HEK293 and Min6 were found to be less than 1%. The Pdx1 Profile is Remarkably Stable Several studies have shown that Pdx1 is modified in response to glucose stimulation and oxidative stress [26,27,28,29]. To test if the endogenous Pdx1 profile is dependent on cellular context or exogenous stimulation we proceeded to investigate the NIA profile of several sources of b-cells. In the embryonic pancreas the developing b-cells can be distinguished from other Pdx1 expressing PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35233