Secondary Lipid Oxidation Products as Modulators of Calpain‑2 Functionality In Vitro

The objective was to understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and, subsequently, to determine the quantity and localization of modification sites. 2-Hexenal and 4-hydroxynonenal incubation significantly decreased calpain-2 activity and slowed the progression of autolysis, while malondialdehyde had minimal impact on calpain-2 activity and autolysis. Specific modification sites were determined with LC-MS/MS, including distinct malondialdehyde modification sites on the calpain-2 catalytic and regulatory subunits. 2-Hexenal modification sites were observed on the calpain-2 catalytic subunit. Intact protein mass analysis with MALDI-MS revealed that a significant number of modifications on the calpain-2 catalytic and regulatory subunits are likely to exist. These observations confirm that specific lipid oxidation products modify calpain-2 and may affect the calpain-2 functionality. The results of these novel experiments have implications for healthy tissue metabolism, skeletal muscle growth, and post-mortem meat tenderness development.