Nanoprobe-based immobilized metal affinity chromatography for sensitive and complementary enrichment of multiply phosphorylated peptides.

Magnetic nanoparticles (MNP, <100 nm) have rapidly evolved as sensitive affinity probes for phosphopeptide enrichment. By taking advantage of the easy magnetic separation and flexible surface modification of the MNP, we developed a surface-blocked, nanoprobe-based immobilized metal ion affinity chromatography (NB-IMAC) method for the enhanced purification of multiply phosphorylated peptides. The NB-IMAC method allowed rapid and specific one-step enrichment by blocking the surface of titanium (IV) ion-charged nitrilotriacetic acid-conjugated MNP (Ti⁴-NTA-PEG@MNP) with low molecular weight polyethylene glycol. The MNP demonstrated highly sensitive and unbiased extraction of both mono- and multiply phosphorylated peptides from diluted β-casein (2 × 10⁻¹⁰  M). Without chemical derivation or fractionation, 1283 phosphopeptides were identified from 400 μg of Raji B cells with 80% purification specificity. We also showed the first systematic comparison on the particle size effect between nano-sclae IMAC and micro-scale IMAC. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis revealed that MNP had a 4.6-fold higher capacity for metal ions per unit weight than did the magnetic micro-sized particle (MMP, 2-10 μm), resulting in the identification of more phosphopeptides as well as a higher percentage of multiply phosphorylated peptides (31%) at the proteome scale. Furthermore, NB-IMAC complements chromatography-based IMAC and TiO₂ methods because <13% of mono- and 12% of multiply phosphorylated peptide identifications overlapped among the 2700 phosphopeptides identified by the three methods. Notably, the number of multiply phosphorylated peptides was enriched twofold and threefold by NB-IMAC relative to micro-scale IMAC and TiO₂, respectively. NB-IMAC is an innovative material for increasing the identification coverage in phosphoproteomics.