is the most abundant phosphorylation on Pdx1, and that it is not regulated by glucose in mouse islets. Materials and Methods Nanofluidic Proteomic Immunoassay (NIA) This assay, here designated NIA, was developed by Cell Bioscience [42]. All runs were done on the Nanopro1000 instrument and performed essentially as described by the manufacturer (http:// www.cellbiosciences.com/nanopro.html). The NIA assay is a fully automated, nanocapillary-based immunoassay based on isoelectric focusing (IEF) to separate the proteins in a regular cell or tissue lysate. Following separation the proteins are linked to the capillary wall through UV irradiation and then probed with primary and secondary antibodies. The secondary antibody is HRP-labeled, which enables sensitive chemiluminescence detection. The signal, which is black dots equivalent to that obtained by western blotting are then analyzed by special software and can be viewed as either a graph or a virtual gel. All NIA data presented in this study were obtained by duplicate analysis of the described lysates in either HNG buffer (20 mM Hepes, pH7.5, 25 mM NaCl, 10% glycerol) or 8 M urea, both supplemented with 20 mM DTT. This was done to compare the NIA profile under conditions of semi- and full- denaturation. Samples were then mixed 1:1 with a sorbitol based premix, containing five peptide standards with known pI (pI ladder 1 cat.no. 040–644). The same premix was used for all runs and it enables the generation of a pH gradient ranging from 4 to 9 (cat.no. 040–319). To establish the pH gradient, the anolyte were 10 mM phosphoric acid and the catholyte were 100 mM sodium hydroxide. The proteins were focused for 40 min. at 15 mW, and then incubated for 2 hours with primary antibody; 4 times wash with TBST and 1 hour with the secondary HRP conjugated antibody, washed again and lastly, chemiluminescence detection with Super- Signal West Dura from Pirece. Concerning peak identification we observe small variations from run to run. The peak at pI 6.40 could therefore vary from 6.36 to 6.44. However, the relative distance between peaks in the same run was always the same. Dephosphorylation was done in vitro using Lambda phosphatase (NEB, Cat.no. P0753). Cell lysate were diluted 10 times in reaction buffer (50 mM HEPES, 100 mM NaCl, 2 mM DTT, 4 mM MnCl2), split in two and treated with 1 ml (400 units) lambda phosphatase or 1 ml water for 30 min. at 37uC. Afterwards the reactions were diluted once in HNG or 8 M urea, both with 20 mM DTT and analyzed by NIA as described above. MS Analysis All MS experiments were performed on a nanoscale HPLC system (EASY-nLC from Proxeon Biosystems) connected to a hybrid LTQ–Orbitrap Velos (Thermo Fisher Scientific) equipped with a nanoelectrospray source (Proxeon Biosystems). Each peptide sample was auto-sampled and separated in a 15 cm analytical column (75 mm inner diameter) in-house packed with 3- mm C18 beads (Reprosil Pur-AQ, Dr. Maisch) with a 2 h gradient from 5% to 40% acetonitrile in 0.5% acetic acid. The MS instrument was operated in data-dependent mode to automatically switch between full-scan MS and MS/MS acquisition. Survey full- scan MS spectra (from m/z 300–1,700) were acquired in the orbitrap with resolution R = 30,000 at m/z 400 (after accumulation to a ‘target value’ of 1,000,000 in the linear ion trap) using a HCD top10 method. The ten most intense peptide ions with charge states $2 were sequentially isolated to a target value of 50,000 using predictive automatic gain control (pAGC) and fragmented by higher-energy collisional dissociation (HCD) in the octopole collision cell using normalized collision energy of Post-Translational Modification of Pdx1 40%. The ion selection threshold was 5,000 counts for HCD and the maximum allowed ion accumulation times were 500 ms for full scans and 250 ms for HCD. All HCD fragment ion spectra were recorded in the orbitrap with a resolution of 7,500 at m/ z 400. For all full scan measurements a lock-mass ion from ambient air (m/z 445.120025) was used for internal calibration when present, as described [49]. Standard mass spectrometric conditions for all experiments were: spray voltage, 2.2 kV; no sheath and auxiliary gas flow; heated capillary temperature, 200uC; predictive AGC with calibrated scaling factor of about 100, and an S-lens RF level of 60%. Identification of Peptides and Proteins by MaxQuant The data analysis was performed with the MaxQuant software (version 1.0.14.7, www.maxquant.org) as described [50] supported by Mascot (www.matrixscience.com) as the database search engine for peptide identifications. We followed the step-by-step protocol of the MaxQuant software suite [51] to generate MS/MS peak lists that were filtered to contain at most six peaks per 100 Da interval and searched by Mascot (Version 2.2.04) against a concatenated target/decoy (forward and reversed) version of the IPI human database version 3.37 (69.316 forward protein entries). Protein sequences of common contaminants such as human keratins and proteases used were added to the database. The initial mass tolerance in MS mode was set to 7 p.p.m. and MS/MS mass tolerance was 0.02 Da. Cysteine carbamidomethylation was searched as a fixed modification, whereas lysine acetylation, serine/threonine/tyrosine phosphorylation, protein N-acetylation, and oxidized methionine were searched as variable modifications. A maximum of two mis-cleavages was allowed while we required strict tryptic specificity. The resulting Mascot.dat files were loaded into the MaxQuant software together with the raw data for further analysis. To minimize false identifications, all top-scoring peptide assignments made by Mascot were filtered based on previous knowledge of individual peptide mass error. Peptide assignments were statistically evaluated in a Bayesian model on the basis of sequence length and Mascot score. We accepted peptides and proteins with a false discovery rate of less than 1%, estimated on the basis of the number of accepted reverse hits. Chicken Electroporations and FACS Analysis Chicken electroporations was performed as previously described [44]. Wholemount immunefluorescence staining on mouse and chicken embryos were performed as previously described [52]. For OPT analysis embryos were stained and scanned on an OPT scanner 3001 M (Bioptonics, Edinburgh UK) according to manufacturers instructions. For FACS all embryos were electro- porated with 10 mg of pCGIG5 vector containing the gene for cytoplasmic EGFP as well as 10 mg of either empty vector or mouse Pdx1WT. The embryos were harvested 24 hours after electroporation by digestion with 300 ml 1% trypsin per 2 embryos for 10 min. at 37uC, with 3 rounds of gentle pipeting. The trypsin were neutralised with DMEM media containing 10% FBS. After centrifugation at 12006g for 5 min. the cells from 12 embryos were resuspended in app. 7 ml FACSflow buffer and sorted by EGFP fluorescence on a FACSaria instrument (BD Bioscience). The resulting fractions were either EGFP negative, app. 1.5 million cells or EGFP positive, app. 50.000 cells. These fractions were centrifuged and the EGFP negative fractions lysed in 100 ml lysis buffer and the EGFP positive fractions in 4 ml. For details on the lysis buffer please refer to the supplementary information. PLoS ONE | www.plosone.org 12 April 2012 | Volume 7 | Issue 4 | e35233