PAMA–Arg brush-functionalized magnetic composite nanospheres for highly effective enrichment of phosphorylated biomolecules

Highly effective enrichment of phosphorylated biomolecules is vital for an in depth and comprehensive phosphoproteome analysis. Among affinity materials, materials based on the immobilized metal affinity chromatography (IMAC) method are most commonly used in phosphopeptide enrichment. However, the problem of metal cation loss in IMAC materials is still a challenge. In this study, a novel strategy has been developed for both highly effective enrichment of phosphorylated biomolecules and prevention of metal cation loss using Fe3O4/polydopamine/poly(2-aminoethyl methacrylate hydrochloride)/arginine (Fe3O4/PDA/PAMA–Arg) composite nanospheres. The magnetic composite nanospheres were prepared by a facile method, possessing uniform morphology, a high saturation magnetization (45 emu g−1), and a quick magnetic response (within 10 s). Noticeably, abundant guanidyl and amino groups of the PAMA–Arg brushes endowed the nanospheres with extremely high detection sensitivity (0.2 fmol), excellent selectivity (β-casein/BSA = 1 : 500), and high recyclability (5 cycles) in phosphopeptide analysis. Furthermore, the Fe3O4/PDA/PAMA–Arg nanospheres exhibited specific separation capacity towards β-casein or ovalbumin (OVA) in the model protein mixture and remarkably selective enrichment performance for phosphorylated biomolecules in real biological samples (egg white, nonfat milk, and rat brain lysate), showing great potential for trace biological detection and proteomics analysis.