ABSTRACT: Cadmium (Cd2+) is a persistent neurotoxic trace metal, yet the native protein targets and protein assemblies associated with cadmium exposure in brain tissue remain incompletely defined. Here, we developed Cd-IMAC-MS, a non-denaturing workflow that couples Cd2+-charged nitrilotriacetic acid (NTA) affinity enrichment with data-independent acquisition (DIA) proteomics to profile cadmium-associated proteins from rat brain. To distinguish Cd-dependent enrichment from bead-derived background and nonspecific adsorption, the discovery experiment used a 2 × 2 design and double-ratio correction (CH/CL versus BH/BL). Cd-NTA enrichment quantified 2,989 proteins and 37,947 peptides, whereas blank-NTA controls quantified 2,425 proteins and 20,559 peptides. The correction framework resolved 80 high-confidence cadmium-associated candidates, including 57 proteins quantified across both matrices and 23 proteins detected only in Cd-NTA enrichments. STRING and cytoHubba analyses identified ANXA5, ANXA2, TOP2A, and SQSTM1 as consensus network hubs. An independent cadmium exposure cohort further prioritized eight reproducible candidates—CTBP1, IQSEC3, TAGLN2, ANXA2, ANXA1, NUCB1, PDHA1, and USP15—linking cadmium exposure to calcium-responsive membrane organization, mitochondrial metabolism, vesicle trafficking, proteostasis, and inflammatory or stress signaling. UniProt annotation and MIB2 residue-level prediction further indicated metal-binding features in CTBP1, IQSEC3, TAGLN2, and USP15. Collectively, Cd-IMAC-MS provides a scalable strategy for native cadmium-associated protein discovery in complex brain tissue and yields a prioritized resource for mechanistic and translational studies of cadmium neurotoxicity.
Shaoxin Huang 
