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.