Identification of myofibrillar substrates for μ-calpain

Abstract

To identify myofibrillar substrates of μ-calpain under post-mortem conditions, a combination of SDS–PAGE, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used. Purified myofibrils were incubated with μ-calpain under post-mortem-simulated conditions for two or four days at 4 °C. The resulting protein changes were analyzed by SDS–PAGE and 2DE. The μ-calpain-mediated protein changes were identified by peptide-mass mapping using MALDI-TOF MS and revealed that desmin, actin, myosin heavy chain, myosin light chain I, troponin T, tropomyosin α1, tropomyosin α4, thioredoxin and CapZ are all degraded in vitro by μ-calpain. The findings that actin and myosin heavy chain are substrates of μ-calpain were rather surprising, as it has previously been reported that these proteins are resistant to μ-calpain degradation. However, both actin and myosin heavy chain are poor substrates compared with desmin.