Characterization of peptides released from rabbit skeletal muscle troponin-T by μ-calpain under conditions of low temperature and high ionic strength

Rabbit skeletal muscle troponin-T (200 μg ml−1) was incubated with μ-calpain (2 μl ml−1) under conditions of low temperature and high ionic strength for 180 min at 4°C and the peptides released analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Troponin-T was hydrolyzed rapidly with the simultaneous appearance of eight peptides with masses of less than 14 up to 26 kDa. Two peptides produced by 10 min of incubation were electroblotted and analysis of these peptides by N-terminal sequencing and mass spectrometry showed that the principal cleavage sites of μ-calpain on troponin-T were at Thr45–Ala46, Leu69–Met70, Glu220–Lys221 and Asn231–Val232. The peptides present in insufficient quantity for electroblotting were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). Cleavage sites were also identified at Met151–Gly152, Asn188–Ile189, Lys223–Arg224, Arg233–Ala234 and Ala240–Lys241. In general, μ-calpain cleaved bonds containing one hydrophobic amino acid residue and mainly towards the C-terminus of troponin-T.