Design of reversibly charge-changeable rhodamine B modified magnetic nanoparticles to enrich phosphopeptides

In the present study, by employing ethylenediaminetetraacetic acid (EDTA), tetraethylene pentaamine (TEPA), and rhodamine B (Rb), we designed and synthesized a magnetic adsorbent (Fe₃O₄@EDTA@TEPA@Rb) on the basis of reversible charge change of Rb and applied to capture phosphopeptides. Rb existing in open planarized zwitterion form when stimulated by acidic loading buffer adsorbs negative phosphopeptides via electrostatic interaction. Under the stimulation of alkalic eluent, ring-closed structure of Rb is formed to elute the enriched phosphopeptides. TEPA containing rich amino groups is used as a crosslinking agent, which is also protonated in acidic loading buffer to bond phosphopeptides. Then phosphopeptides are eluted when TEPA deprotonates in alkalic eluent. Coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) detection, phosphopeptide signals originated from 0.4 fmol/μL β-casein digests were successfully detected. In addition, Fe₃O₄@EDTA@TEPA@Rb can also efficiently enrich phosphopeptides from skimmed milk, human serum and saliva samples (26, 4, 39 phosphopeptides, respectively), opening a new gallery for phosphopeptides-related analysis. In general, the developed adsorbent has the great potential for further application in the near future.