Post-synthesis of biomimetic chitosan with honeycomb-like structure for sensitive recognition of phosphorylated peptides

•A biomimetic honeycombed affinity chromatography (IMAC) adsorbent was prepared via a facile route for selective enrichment of phosphopeptides.•The post-modification on the surface of the material was performed in lower temperature and ehanced the loading amount of Ti4+ ions.•CTSM@AMPA-Ti4+ shows excellent selectivity and sensitivity as well as size exclusion effect for phosphopeptides.•Good cyclic stability was achieved even for ten rounds of enrichment, and a degree of pH tolerance. Chemical modification of biological materials is indispensable for enrichment of phosphorylated peptides. In this work, we synthesized a biomimetic honeycombed affinity chromatography (IMAC) adsorbent by preparing Crosslinked Chitosan, chelating aminomethyl phosphate decorated with Ti (IV) cation. The as-prepared CTSM@AMPA-Ti4+ composites with stable structure, low steric hindrance, and high Ti4+ loading amount were used as a promising adsorbent for enrichment of phosphopeptides. CTSM@AMPA-Ti4+ showed extremely high sensitivity (0.4 fmol) and selectivity at a low composition molar ratio of β-casein/BSA (1:1000). What's more, it can keep its performance in the case that used to capture phosphorylated peptides from standard protein ten times or soaking in the acid/base solution for a long time. In addition, CTSM@AMPA-Ti4+ successfully captured 35 phosphorylated peptides from human saliva. This study offers a way about diversiform functionalization of CTSM in phosphoproteome analysis and disease research.