for Pdx1 [23]. It is unclear why reductions in Pdx1 protein levels has such deteriorating effects on the b-cell, however, a recent study suggests that it may be linked to mitochondrial dysfunction. Thus preventing opening of the mitochondrial permeability transition pore through deletion of the Peptidyl-prolyl cis-trans isomerase (Ppif) gene restores many of the defects caused by Pdx1 insufficiency [24]. Since even subtle changes in Pdx1 levels dramatically impacts b-cell performance it is generally acknowledged that the biological activity of Pdx1 tightly regulated at the post-translational level. Fitting this hypothesis at least three different post-translational modifications have been reported on Pdx1 including phosphoryla- tion [25,26,27,28,29,30,31,32,33,34,35], glycosylation [36] and sumoylation [37]. Phosphorylation, in particular, has been shown to regulate several important aspects of Pdx1 activity in response to elevated glucose levels, such as nuclear-cytoplasmic shuttling [26,27], DNA binding [27,28,38] and interactions with transcrip- tional co-factors [25,39]. Several different kinases have been shown to phosphorylate Pdx1 and to date five different residues have been suggested to be Pdx1 kinase substrates. Amino acids S61 or S66 are phosphorylated by Glycogen synthase kinase 3 (Gsk3) and ERK1/2 [29,31] and this modification targets Pdx1 for proteasomal degradation in response to oxidative stress [29]. Amino acids S268 and S272 (S269 and S273 in mouse) are phosphorylated by Hipk2 [40] or in response to low glucose [30] and T11 is phosphorylated by DNA-dependent kinase in response to DNA damage [33]. Yet, despite the attention several important questions remain unan- swered. For example it is unclear to what extent Pdx1 is modified under normal conditions? How many PTMs are present at a given time? How do different PTMs influence each other? How is the endogenous Pdx1 modified in respond to various treatments? Here we have employed a novel technique called nanofluidic proteomic immunoassay (NIA) [41,42] to study post-translation- al modifications of Pdx1 in various cell types. NIA employs isoelectric focusing to separate differently charged protein species of the same protein which is then detected using a pan-specific antibody. A particular advantage of this technology is that experiments can be carried out from scarce samples [42]. We have therefore been able to identify and study different protein species of endogenous Pdx1 in cell lines, during development and in mature b-cells. In eukaryotic cells we observe that the Pdx1 protein separates into four peaks, while Pdx1 purified from bacteria only produces a single peak. There is no apparent difference in SDS-PAGE mobility between mammalian and bacterial Pdx1 proteins and we therefore conclude that the mammalian profile is the result of post- translational modifications. Treating the Pdx1 protein with phosphatase revealed the presence of one primary phosphory- lation which was mapped to serine 61 using both alanine scanning and mass spectrometry. Remarkably, Pdx1 protein obtained from, embryonic tissue, adult pancreas, isolated islets, bTC cells, Min6 cells and from Pdx1 over-expressed in aTC, L and HEK293 cells produces almost identical profiles. Since the single phosphorylation is present in all cells and tissues tested we speculate that it is mediated by a ubiquitously expressed kinase rather than a specific signaling pathway. In line with this we were unable to demonstrate any alterations in the Pdx1 profile following high or low glucose treatment of mouse islets in culture. And although we confirm that Pdx1 is phosphorylated during embryonic pancreas development, we find that Pdx1S61A is fully capable of inducing ectopic Nkx6.1 expression and budding of pancreatic like structures. Post-Translational Modification of Pdx1 Results Over Expressed Pdx1 Appear as Multiple Protein Species To investigate the extent of post-translational modifications on the endogenous Pdx1 protein we applied a novel technology called NIA [41,42]. Isoelectric focusing is used to separate differently charged species of a protein which subsequently can be detected using a pan-specific antibody that recognizes these species with similar affinity (assuming equal antibody binding to the different protein species). The raw data can be examined as bands on a gel or as peaks (Fig. 1A) where the relative abundance of the different protein species can be estimated from the height of the peaks or as the area under the curves (Fig. 1B). To precisely determine which peaks can be assigned to Pdx1 we performed western blot and NIA analysis on lysates from cells over-expressing wild type mouse Pdx1 (Pdx1WT), using two different antibodies against Pdx1. A polyclonal goat-a-Pdx1 antibody and a monoclonal mouse-a-Pdx1 antibody [43]. When used for western we found that both antibodies specifically detected a 40 kDa band in cells transfected with Pdx1WT (Fig. 1C). When the same lysates was used for NIA the empty vector control displayed no peaks whereas both antibodies produced peaks at pI 5.9, 6.0, 6.1, 6.3 and 6.4 in Pdx1 transfected cells (Fig. 1D and E). The only difference was the 5.8 peak which is absent when using the mouse antibody. Pdx1 is Subject to Post-translational Modification in Mammalian Cells To test if the antibodies specifically recognize various protein species of Pdx1 the entire profile should be shifted if the overall pI is changed. We therefore added a negatively charged 3xFLAG epitope tag to the C-terminus of wild type Pdx1 (Pdx13xFLAG). This changes the predicted pI of Pdx1 from 6.4 for the wild type to 5.5 for the 3xFLAG tagged Pdx1. The increase in size was confirmed on a western blot (Fig. 2A) and as predicted the NIA profile of FLAG tagged Pdx1 was shifted to the left (Fig. 2B; red line). Importantly, all peaks are shifted to the left indicating that the added FLAG tag affects all Pdx1 protein species. It should be noted that the leftward shift of the profile is associated with a compression of the NIA profile, leading to the 6.0 and 6.1 as well as the 6.3 and 6.4 peaks becoming indistinguishable. However, to confirm that Pdx1 is actually modified we went on to purify Pdx1 from either bacteria or mammalian cells and analyzed the purified proteins using SDS-PAGE and NIA. The cDNA encoding wild type Pdx1 was cloned into two vectors which allowed expression in either bacteria or mammalian cells, respectively. Pdx1 protein was subsequently purified using the mouse-a-Pdx1 antibody for immunoprecipitation (IP) and then the goat-a-Pdx1 antibody for western and NIA. On SDS-PAGE the purified protein migrated at approximately 40 kDa and could be detected by western from both bacteria and HEK293 cells while control samples where negative (Fig. 2C). When the same samples were analyzed on NIA bacterial Pdx1 gave a single peak at pI 7.2 (Fig. 2D) while the profile obtained from mammalian Pdx1 was similar to that obtained without IP and the most prominent peaks were detected at pI 5.9, 6.0, 6.1, 6.4 but we also observed lower intensity peaks at pI 5.6, 6.5, 6.9 and 7.2 (Fig. 2E). To further validate these results we repeated the experiment using the Pdx13xFLAG construct. When this approach was used to purify protein from bacteria we detected some unspecific bands both using coomassie and western blotting (Fig. 2F), importantly, the Pdx13xFLAG band was the strongest signal in the lane and the controls were blank. NIA analysis of Pdx13xFLAG obtained from bacteria revealed one Pdx1 specific peak at pI 5.9, while a smaller 5.6 peak was present in both Pdx1 and control lysates (Fig. 2G). We therefore believe that the PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35233