Preparation Of Novel Magnetic Adsorbents For High-performative Capture Of Phosphopeptides

Phosphorylated proteins maintain the normal metabolic activities of the organism.Accurate detection of phosphopeptides is of great significance.Phosphopeptides are difficult to be directly recognized by matrix-assisted laser desorption ionization time-of-flight mass spectrometer（MALDI-TOF-MS）due to the inherent characteristics of low abundance,difficult ionization and interferences of complicated biological fluids.It is very necessary to develop strategies to enrich phosphopeptides.In this study,based on electrostatic affinity chromatography,four adsorbents for enriching phosphopeptides were developed.Details about the four adsorbents are as follows:The interconversion between zwitterion and closed-loop lactone forms of rhodamine B（Rb）corresponds to the enrichment-elution of phosphopeptides.However,Rb-based adsorbents are rarely studied.With ethylenediamine tetraacetic acid（EDTA）and tetraethylenepentamine（TEPA）as crosslinking agents,Rb modified magnetic adsorbent was synthesized（Fe₃O₄@EDTA@TEPA@Rb）.After the modification of 3-[2-（2-Aminoethylamino）ethylamino]propyl-trimethoxysilane（AAAPTES）and Rb,a novel adsorbent（Fe₃O₄@NH₂@Rb）was synthesized.Stimulated by acidic loading buffer,Fe₃O₄@NH₂@Rb with rich positive charges accurately capture negatively charged phosphopeptides.After shaking with eluent,deprotonated AAAPTES and Rb existing in closed-loop lactone form no longer interact with phosphopeptides,that is,phosphopeptides are eluted.Based on the p H response of FITC,with polyethyleneimine（PEI）as the cross-linking agent,a magnetic adsorbent modified with FITC（Fe₃O₄@PEI@FITC）was synthesized by the interaction between isothiocyano and amino groups.Highly protonated PEI and cationic FITC have strong interaction with electronegative phosphopeptides.Based on the principle of self-assembly,a magnetic adsorbent（Fe₃O₄@NE@PL）coated with norepinephrine（NE）andε-polylysine（PL）was synthesized by mechanical stirring in water at room temperature.Under the combined action of intermolecular force and electrostatic force,Fe₃O₄@NE@PL can efficiently attract hydrophilic phosphopeptides.The developed methods of Fe₃O₄@EDTA@TEPA@Rb,Fe₃O₄@NH₂@Rb,Fe₃O₄@PEI@FITC and Fe₃O₄@NE@PL for detecting phosphopeptides have low detection limit,high selectivity,strong stability and good cyclicity,and successfully enrich phosphopeptides from complex biological samples.Generally speaking,the four adsorbents open a new gallery for the analysis of phosphopeptides,and they are likely to be further used in the future.