RESUMO
As one of the most common post-translational modification of proteins, protein phosphorylation plays a vital role in many physiological processes. The enrichment of phosphopeptides is highly important before the mass spectrometry detection since phosphopeptides are susceptible to interferences from high-abundance non-phosphopeptides. In this study, we designed a novel magnetic composite (Fe3O4@PDA-PEI-Fe3+) for phosphopeptide enrichment with a facile protocol. The developed Fe3O4@PDA-PEI-Fe3+ is a marvelous material with multiple functional groups, and can effectively enrich phosphopeptides through the synergistic effect of three mechanisms, i.e., immobilized metal ion affinity chromatography raised form Fe3+, electrostatic interaction between amine and phosphate groups, and hydrogen bond between the hydrogen atoms of amine groups and oxygen atoms of phosphate groups. Combined with mass spectrometry, the material shows excellent enrichment performance, high sensitivity (0.4 fmol), good selectivity (ß-casein:BSA= 1:500, w:w), and stable reusability (at least 5 cycles). In addition, the material was successfully applied to enrich phosphopeptides from skim milk and human saliva samples, implying that it is an ideal adsorbent for the phosphopeptide enrichment in complex biological samples and provides valuable insights into the field of phosphopeptide analysis.