The serine/threonine kinase protein kinase C epsilon (PKC epsilon) has been shown to be a critical component in the heart's resistance to cell death following ischemic insult. Recent studies have indicated that PKC epsilon forms multi-protein signaling complexes to accomplish signal transduction in cardiac protection. Using two-dimensional electrophoresis (2DE), combined with matrix-assisted laser desorption ionization mass spectrometry (MS), the initial analysis of these complexes identified signaling molecules, structural proteins, and stress-activated proteins. The initial analysis, although fruitful, was limited by the number of proteins revealed on the 2D gels. It was also apparent that many known cardiac protective functions of PKC epsilon could not be fully accounted for by the proteins identified in the initial analysis. Here we reported the identification of an additional 57 proteins in PKC epsilon complexes using complimentary separation techniques, combined with high sensitivity MS. These techniques include 2DE or large format 1D SDS-PAGE followed by LC/MS/MS and solution trypsin digestion followed by LC/MS/MS, all of which yielded novel data regarding PKC epsilon protein complexes. Nanoscale LC/MS/MS for the analysis of gel-isolated proteins was performed with sub-femtomole sensitivity. In contrast to 2DE analyses, the identification of proteins from 1D gels was independent of their visualization via staining and allowed for the identification of proteins with high isoelectric points. We found that PKC epsilon complexes contain numerous structural and signaling molecules that had escaped detection by our previous analyses. Most importantly, we identified two new groups of proteins that were previously unrecognized as components of the PKC epsilon complex: metabolism-related proteins and transcription/translation-related proteins.