Cd-IMAC-MS Enables Exploratory Profiling of Cadmium-Associated Proteins in Rat Brain after Cadmium Exposure

Cadmium (Cd²⁺) is a persistent neurotoxic metal, but the brain proteins recoverable by cadmium-affinity enrichment after exposure remain incompletely defined. We developed cadmium-immobilized metal affinity chromatography coupled with mass spectrometry (Cd-IMAC-MS), which combines non-denaturing Cd²⁺-charged nitrilotriacetic acid (Cd-NTA) affinity recovery, blank-NTA controls, and data-independent acquisition (DIA) proteomics. The discovery experiment comprised 12 independent animals in a balanced 2 × 2 exposure-by-affinity-matrix design (n = 3 per cell). For 2,119 proteins quantified across all four cells, an ordinary least-squares interaction screen identified 26 nominal candidates (10 positive and 16 negative interaction effects); none met Benjamini-Hochberg-adjusted q < 0.05. A separate Cd-NTA-only CH - CL branch identified 23 nominal candidates (six positive and 17 negative effects), yielding a 49-protein exploratory union; no protein met q < 0.05 after pooled adjustment across 2,988 discovery tests. In a lower-dose prefrontal-cortex cohort, 775 of 8,209 proteins met nominal P < 0.05 and none met q < 0.05. Six symbols overlapped the discovery set, and CTBP1 and PDHA1 also occurred in the Comparative Toxicogenomics Database list. Because no standalone sample-ID-to-treatment key was available, the reported meanCon-meanCase signs were not assigned an exposure direction. The functional-context analysis, protein-association network, database annotations, and residue-level predictions are interpreted as secondary, hypothesis-generating evidence. Cd-IMAC-MS therefore prioritizes candidates for orthogonal binding and functional validation rather than establishing direct in vivo Cd²⁺ binding.