Top‐Down Proteomics Study of Aging and Sexual Dimorphism of Zebrafish Brains

Aging significantly impacts brain function and increases susceptibility to neurodegenerative diseases, with notable sex dimorphism observed in aging-related dementia, such as Alzheimer's disease. To better understand the molecular mechanisms of aging and dementia, it is essential to globally and accurately characterize biomolecules (e.g., proteins) in the brain as a function of age and sex. Here, we present one of the first studies of aging and sexual dimorphism in brains using the zebrafish (Danio rerio) model, employing mass spectrometry (MS)-based quantitative top-down proteomics (TDP). We analyzed proteoforms in male and female zebrafish brains across three ages (6, 16, and 24 months) using capillary zone electrophoresis-tandem MS (CZE-MS/MS). We revealed significant sex- and age-related differences in the abundance of key proteoforms, including those associated with neurofilament assembly, dopaminergic neuron differentiation, and synaptic vesicle priming. Notably, we identified a truncated proteoform of AP2B1, a subunit of the AP2 adaptor complex closely associated with clathrin-mediated endocytosis and cellular aging, in female zebrafish aged 24 months exclusively, suggesting potential age- and sex-specific roles in brain aging. Additionally, significant changes were observed in proteoforms involved in energy metabolism, structural maintenance, and neurotransmitter release, providing a new opportunity for a better understanding of the molecular mechanisms of brain aging and sexual dimorphism. These findings highlight the effectiveness of CZE-MS/MS in TDP for identifying and quantifying proteoforms, offering a deep view of sex-specific proteoform dynamics during brain aging.