Neuroglobin is an ancient protein that is more related to the annelid Aphrodite

To validate the efficacy of phospho protein enrichment, 40 ug of proteins from total cellular lysate, elution fraction containing the highly concen trated and purified phosphoproteins, and flow through fraction were separated by 6% SDS PAGE, followed by Western blotting using anti phosphotyrosine antibody. The concentration of the phosphopro [You must be registered and logged in to see this link.] teins was determined using a 2 D Quantification Kit. Protein labeling Phosphoproteins from the elution fractions were preci pitated using chloroform methanol as described by Wessel and Flugge, resolubilized in 2D DIGE sam ple buffer, and adjusted to pH 8. 5. Equal amount phosphoproteins from six samples were pooled together as the internal standard. Three EGF stimulated samples and three EGF unstimu lated samples were randomly labeled with Cy3 or Cy5, while internal standards were labeled with Cy2, using 200 pmol fluorochrome25 ug protein.

Labeling reactions were performed on ice in the dark [You must be registered and logged in to see this link.] for 30 min, and then quenched by the addition of 1 uL 10 mM lysine for 10 min. 2D DIGE Cy3 and Cy5 labelled samples from each pair of EGF treated and untreated cells were combined before mixing with 25 ug Cy2 labelled internal standards. An equal volume of 2 sample buffer was added to the sample and the total volume was made up to 450 uL with rehydration buffer. The samples were applied to IPG strips and focused on an IPGphor. The focused IPG strips were equilibrated, and then were transferred to the tops of 12. 5% polyacrylamide gels and run for about 7 h, using low fluorescence glass plates on an Ettan DALT II sys tem.

All electrophoresis proce dures were performed in the dark. The biological triplicate EGF stimulated and unstimulated cells and the internal standard were run on three gels as analytic gels. In addition, we performed another strip in parallel as a preparative [You must be registered and logged in to see this link.] gel for spot pickings, as described above, except that the IPG strip was loaded with 1000 ug pro teins and the gel was stained with Coomassie brilliant blue. After SDS PAGE, the three analytic gels were scanned on a Typhoon 9410 scanner at appropriate excitationemission wavelengths specific for Cy2, Cy3 and Cy5, to generate nine protein spot maps. Image analysis Images were cropped using ImageQuant TL 2005 soft ware from 2D DIGE gels, and analyzed using DeCyder 6. 5 software according to the manufacturers recommendations.

The DeCyder differential in gel analysis module was used for pairwise comparisons of each EGF stimulated and unsti mulated cell sample to the internal standard in each gel. The DeCyder biological variation analysis module was then used to simultaneously match all nine protein spot maps and, using the Cy3Cy2 and Cy5Cy2 DIA ratios, to calculate average abundance changes and paired Students t test p values for the variance of these ratios for each protein pair across all samples. The dif ferential protein spots that altered consistently in all nine protein spot maps were selected for identification. Protein identification by MS All the differential phosphoprotein spots were excised from stained preparative gels using punch, destained, and in gel trypsin digestion was performed as previously described by us.