Hands-free phosphoproteomics workflow with a high-throughput automated system enabled by superhydrophilic...

We report the fabrication of superhydrophilic polymer-functionalized magnetic nanoparticles, Fe₃O₄@GMAG@NH₂@PO₃@Ti⁴⁺ (denoted as Ti⁴⁺-MagBeads), which are suitable for both SP3-based on-bead protein digestion and subsequent Ti⁴⁺-IMAC-based phosphopeptide enrichment in an integrated and automated workflow. The dextran-based polymer layer prepared via atom transfer radical polymerization provides abundant Ti⁴⁺ chelation sites while effectively suppressing nonspecific adsorption. In addition, the highly hydrophilic surface promotes protein immobilization under organic solvent-induced SP3 conditions, enabling seamless integration of on-bead digestion and phosphopeptide enrichment in an automation-compatible workflow. The Ti⁴⁺-MagBeads exhibit robust enrichment performance, including high selectivity (α-casein/BSA = 1:1000), high sensitivity (0.5 fmol), and high recovery (> 90%). Using the Orbitrap Astral Zoom/DIA platform, Ti⁴⁺-MagBeads enabled the identification of more than 5,000 phosphoproteins and over 31,000 phosphopeptides from as little as 1 µg of HeLa peptide input. These results demonstrate the applicability of the workflow to low-input phosphoproteomic analysis when coupled with high-sensitivity Orbitrap Astral/DIA acquisition. Furthermore, automated phosphoproteomic analysis of PD-1 antibody-treated rat subcutaneous melanoma FFPE tissues provided an initial assessment of treatment-associated phosphorylation changes, supporting the feasibility of this workflow for retrospective FFPE-based phosphoproteomic studies. Taken together, these results suggest that Ti⁴⁺-MagBeads offer a streamlined, integrated, and automation-compatible strategy for phosphopeptide enrichment and low-input FFPE-based phosphoproteomic analysis.