Dynamic dose-time proteomic profiling of cadmium exposure in rat prefrontal cortex

Cadmium (Cd) is a well-recognized neurotoxic metal whose effects on the central nervous system accumulate with both exposure intensity and duration. However, how dose and time jointly sculpt brain proteome trajectories-and which early molecular events emerge under low-dose, short-duration exposure-remain insufficiently resolved. Time-resolved proteomics can map these trajectories, but requires explicit dose-time deconvolution to separate main effects from interaction-driven changes while accounting for biological heterogeneity.

We conducted a 3 × 3 oral-exposure design in Sprague-Dawley rats (vehicle control, CdCl2 30 μg/kg/day and 1 mg/kg/day; 2/4/8 weeks) and profiled the prefrontal cortex using deep DIA (diaPASEF) proteomics. An integrative pipeline combined Mfuzz (soft trend clustering), STEM (short time-series pattern discovery), and linear mixed-effects modeling (LMM) with an explicit Dose×Time interaction to deconvolve main and interaction effects while adjusting for sex, baseline body weight, and batch. Functional context was derived from GO/KEGG enrichment and protein-protein interaction (PPI) analyses with FDR control.

After QC, 8209 proteins were quantified. LMM identified 173 proteins significant for the Dose main effect, 476 proteins significant for the Time main effect, and 356 proteins significant for the Dose×Time interaction term (BH-FDR q < 0.05). Across the coefficient matrix, Dose×Time coefficients frequently exceeded the corresponding main-effect coefficients in magnitude, indicating substantial interaction-associated structure in cadmium-responsive proteome variation. Cross-method trend analyses converged on Itpr1 and Pde5a as early-response candidates, each down-regulated at 2 weeks/30 μg/kg and accompanied by coherent shifts in Ca²⁺ handling (e.g., Camk2a/b, Atp2b3, Slc8a1) and glutamatergic receptors (e.g., Grin2d, Gria3/4). Enrichment highlighted network-level perturbations spanning calcium signaling, cGMP-PKG, and glutamatergic synapse (e.g., synaptic membrane, q = 2.1 × 10⁻⁵).

By explicitly deconvolving dose, time, and their interaction, we map dynamic proteome responses in the prefrontal cortex and highlight an early Itpr1/Pde5a-centered calcium-cGMP-glutamatergic signature that is detectable under low-dose, short-duration exposure. The accompanying DIA dataset and analytical workflow provide a reusable resource for mechanistic hypothesis generation and candidate readout nomination across neurotoxicants.