Highly Efficient Separation, Enrichment, and Recovery of Peptides by Silica-Supported Polyethylenimine

Highly efficient and charge-selective adsorption and desorption of peptides at trace level by a solid-phase adsorbent is described. The adsorbent of SiO2@PEI is synthesized by covalent immobilization of branched polyethylenimines (PEI) exclusively on the outer surface of the porous silica particles (∼300 μm). For aqueous peptides (Mw = 600–3000 Da), SiO2@PEI can capture the negatively charged ones and leave the positively charged ones intact, and by adjusting pH of the system peptides with different isoelectric points (pIs) can be well separated. Targeted peptide at low abundance (at least as low as 0.1 mol % with respect to the highest one) can be well separated. The association constants of K > 1012 M–1 at pH > pI and K < 104 M–1 at pH < pI are found; that is, selectivity > 108 is generally available. Thus, a peptide even at sub-femtomolar level can be extracted and eluted for analysis, and efficient recovery (79–92%) of the peptides is found. The extraction is mainly promoted by multisite electrostatic interaction, and the hydrophilic and cationic properties of PEI at low pH play a unique role in desorption efficiency and selectivity. The unbiased nature of this method renders the adsorbent applicable to the efficient separation of a broad spectrum of peptides, including those with similar pIs.