Preparation of Core‐Shell Structured Magnetic Superhydrophilic Extractant for Enrichment of Phosphopeptides

Abstract

Protein phosphorylation regulates and controls protein activity and function, which is the most basic and important part in organisms’ lives. However, the low abundance and ionization inhibition of phosphopeptides make it difficult to understand and analyze the protein phosphorylation mechanism. In this study, we synthesize a core‐shell structured magnetic Ti4+‐based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. The introduction of negatively charged carbon layer increases the specific surface area and avoids loss of the immobilized Ti4+ during sample loading and washing. The synthesized magnetic superhydrophilic extractant is employed to separate and enrich phosphopeptides from standard protein digests coupled with MALDI‐TOF MS. It exhibits excellent enrichment performance of high selectivity (1 : 2000 molar ratio of β‐casein and BSA), sensitivity (low detection limit of 4 fmol) and preferable reusability (at least 12 times) as well as excellent adsorption capacity of 142.5 mg ⋅ g−1 and high recovery rate of 85.6 %. Moreover, sixteen phosphopeptides and four endogenous phosphopeptides can be accurately identified from non‐fat milk and human serum, further indicating the great potential and bright application in phosphoproteomics analysis.