Trinity synergy of guanidine-functionalized polymer brush-modified β-cyclodextrin polymer magnetic beads for recognition of phosphopeptides in serum of Alzheimer's disease.

Systematic profiling of protein phosphorylation is pivotal for elucidating the molecular pathogenesis of Alzheimer's disease (AD). Here, we report the rational design of ‌magCEP@PEI@G‌, a multifunctional magnetic adsorbent integrating β-cyclodextrin-epichlorohydrin polymers, polyethyleneimine (PEI), and guanidino functional groups to enable synergistic phosphopeptide enrichment. This hybrid material achieves unprecedented performance: ‌10-cycle reusability‌ with >95% retention of enrichment efficiency, an ultra-low limit of detection (‌0.2 fmol‌), exceptional selectivity (‌5000:1‌ phospho/non-phospho peptide ratio), rapid elution (‌10 min‌), and near-perfect recovery (‌105.0 ± 0.5%‌). Applied to serum samples from three AD patients, magCEP@PEI@G enabled the robust identification of ‌135 unique phosphopeptides. These results establish magCEP@PEI@G as a highly efficient, scalable platform for the sensitive enrichment of low-abundance phosphopeptides and hold substantial promise for the ‌early diagnosis of Alzheimer's disease‌.