FIGURE 1.

Schematic flow of a gel-based protease proteomic study to identify putative calpain substrates. We developed a gel-based protease proteomic strategy for identifying putative calpain substrates. After IEF separation, immobilized proteins on DryStrips were incubated with (A) or without (B) purified recombinant m-calpain followed by separation by SDS-PAGE. Altered protein spots in Coomassie Brilliant Blue G-250-stained gels were subjected to in-gel digestion followed by MALDI-TOF mass spectrometry. Lists of putative calpain substrates were made after searching through databases as described in the text. Using both culture and rat brain disease models, the identified candidates were subjected to further validation as calpain substrates.