Free-standing lamellar 3D architectures assembled from chitosan as a reusable titanium-immobilized affinity membrane for efficiently capturing phosphopeptides

Protein phosphorylation is involved in many biological processes and associated with some diseases. However, because of the low abundance and large dynamic changes of phosphopeptides in biological samples, it is necessary to enrich phosphopeptides before mass spectrometry detection. Many kinds of materials have been developed for phosphopeptide enrichment, but most of them are disposable and their preparation processes are toxic. In this study, a green strategy was presented to fabricate immobilized metal affinity chromatography (IMAC) materials. Using chitosan as a precursor, polyethylene glycol diglycidyl ether as a cross-linking agent and 2,3,4-trihydroxybenzaldehyde as a functional monomer, a series of chitosan membranes (CMs) with free-standing lamellar 3D architectures were prepared via a freeze-casting technique and after chelating with titanium ions (Ti4+) could serve as IMAC adsorbents for phosphopeptide enrichment in biological samples. Due to the stronger chelating force between Ti4+ and the pyrogallol ligand than that between Ti4+ and the phosphate groups, the IMAC material is reusable for the enrichment of phosphopeptides, exhibiting an excellent enrichment performance and a higher capacity than other IMAC materials such as organosilica hybrid monolith and carbonaceous spheres. Besides, four endogenous phosphopeptides and two dephosphorylated fragments were captured by Ti4+-CM from the human serum. Therefore, Ti4+-CM demonstrates potential commercialization prospects because of its green preparation process and reusability.

Automated summary

A green strategy was presented to fabricate IMAC materials for phosphopeptide enrichment, showing excellent performance and higher capacity compared to other IMAC materials. Ti4+-CM successfully captured multiple endogenous phosphopeptides from human serum, indicating potential commercialization prospects due to its green preparation process and reusability.