Performance of nondenaturing micro 2-DE followed by third-dimension SDS-PAGE in the analysis of Escherichia coli soluble proteins

In a previous paper, we reported on the analysis of Escherichia coli (strain K‐12) soluble proteins by nondenaturing micro 2‐DE/3‐DE and MALDI‐MS‐PMF Manabe, T., Jin, Y., Electrophoresis 2010, 31, 2740–2748]. To evaluate the performance of the 2‐DE/3‐DE technique, a nondenaturing 2‐DE gel just after the second‐dimension run was cut into 12 vertical strips, each 2 mm‐wide strip was set on a micro slab gel, and third‐dimension SDS‐PAGE was run in parallel. Each of the twelve 3‐DE gels showed about 150–200 CBB‐stained spots. Two of the 3‐DE gels were selected for the assignment of polypeptides using MALDI‐MS‐PMF and totally 161 polypeptides were assigned on the two 3‐DE gels, in which 81 have been assigned on the nondenaturing micro 2‐DE gel and 80 were newly assigned. Most of the newly assigned polypeptides resided in faintly stained spots on the 3‐DE gels, which indicates that the polypeptides were purified in the process of the third‐dimension separation. The comparisons of the apparent mass values estimated from the second‐dimension (nondenaturing pore‐gradient PAGE) mobility with those estimated from the third‐dimension (SDS‐PAGE) mobility suggested the oligomer structures of the assigned polypeptides and they matched well with those described in a database (UniProtKnowledgebase). The technique of nondenaturing micro 2‐DE/3‐DE, combined with MALDI‐MS‐PMF, could become an efficient method to obtain information on the quaternary structures of hundreds of cellular soluble proteins simultaneously because of its high efficiency in protein/polypeptide separation and assignment.