Design of a novel rhodamine B based pH-responsive magnetic adsorbent and its application in phosphopeptidomics

Protein phosphorylation is one of the most critical post-translational modifications for regulating protein activity and function. Direct mass spectrometry (MS) analysis of phosphopeptides presents significant challenges due to their inherently low abundance and the inefficient ionization characteristics. In this study, we report the design of a novel pH-responsive magnetic adsorbent, denoted as Fe₃O₄@NH₂@Rb, for highly selective capture of phosphopeptides. This innovative material leverages dynamic and reversible electrostatic interactions between bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane (Bis) and rhodamine B (Rb), which are responsive to pH changes. Under acidic conditions, Bis undergoes protonation while Rb transforms into a ring-opening zwitterionic form. The rapid and reversible transformation of Bis and Rb facilitates the efficient adsorption under acidic conditions and elution in alkaline environments. As anticipated, Fe₃O₄@NH₂@Rb exhibits high enrichment performance for phosphopeptides from both standard protein digests and complex biological samples. This advancement holds great potential to significantly contribute to the phosphoproteomics research.