Redox Stoichiometry at the Single-Residue Level Using Mass Spectrometry Reveals Dynamic Methionine Sulfoxide Speciation in Actin and Calmodulin during Brain Aging

Methionine oxidation to methionine sulfoxide (MSox) is often viewed as a nonspecific modification from reactive oxygen species. However, oxidation at specific methionine sites, such as Met44/47 in actin and Met77 in calmodulin, can be reversed by methionine sulfoxide reductase (Msr) and other enzyme families. This study uses liquid chromatography coupled with mass spectrometry to comprehensively investigate actin and calmodulin-based MSox speciation within the mouse hippocampus in an Alzheimer's disease (AD) model (5XFAD), reflecting neuroinflammation and oxidative stress. Concurrent detection of both oxidized and unmodified peptides enabled direct calculation of absolute oxidation stoichiometry and protein-normalized % occupancy ─an analytical dimension seldom attainable for most post-translational modification studies. Our results indicate age-dependent but not AD-dependent redox dynamics. In actin, D-loop Met44/47 declined from ∼9 to ∼5% between 3 and 6 months and then rose to ∼14% by 9 months, while H-loop Met269 remained stable at ∼5% MSox. In calmodulin, linker Met77 climbed steadily with age (but not AD), whereas C-lobe Met145/146 fell sharply from 20 to ∼8% MSox from 3 to 9 months. These findings highlight dynamic, age-related methionine oxidation patterns in actin and calmodulin within the mouse hippocampus, likely relevant to brain development and aging.