Arginine-rich protamine modified magnetic microspheres for efficient enrichment of phosphopeptides.

The low abundance of phosphorylated proteins in actual samples, the complexity of biological specimen matrices, and the scarcity of high-specificity affinity materials have posed a persistent challenge in achieving highly selective and efficient capture of phosphopeptides. Herein, a novel strongly positively charged arginine-rich material was designed for effective enrichment of phosphopeptides by simply cross-linking protamine onto magnetic microspheres (denoted as FeO@PTA). The FeO@PTA microspheres possessed regular mesoporous structure, excellent thermal stability, and superior magnetic responsiveness, resulting in satisfactory performance in phosphopeptide enrichment with high selectivity (β-casein:BSA = 1:1000), high detection sensitivity (detection limit of 1.0 fmol μL), and good reusability (at least 5 times). Molecular docking simulations demonstrated the enrichment mechanism and binding affinity between protamine and phosphate. In addition, the results of phosphopeptide capture from tryptic digest of nonfat milk, human serum and saliva samples (n = 3) demonstrated the great potential of FeO@PTA in identification of low-abundance phosphopeptides in biological samples.