Ti4+-immobilized chitosan-coated magnetic graphene oxide for the highly selective enrichment of phosphopeptides

Abstract

For in-depth analysis of phosphorylated proteomics, the highly sensitive and selective capture of phosphopeptides from intricate biological samples is extremely significant. In this work, a late-model immobilized metal ion affinity chromatography (IMAC) material was fabricated and applied to enriching phosphopeptides from biological samples by abundant Ti⁴⁺ ions, which were chelated to crosslinked chitosan (CS) loaded on the surface of magnetic graphene oxide (GO) (denoted as mag GO–CS–Ti⁴⁺). The high amount of crosslinked CS endowed the IMAC material with highly hydrophilic properties and abundant active sites for Ti⁴⁺ ions on the surface. These characteristics ensure the great performance of mag GO–CS–Ti⁴⁺ in the selective enrichment of phosphopeptides from the tryptic digest of β-casein with high selectivity (phosphopeptides to non-phosphopeptides at a molar ratio of 1 : 400), high sensitivity (0.5 fmol), large enrichment capacity (66.6 mg g⁻¹), prominent phosphopeptide recovery (93.11%), and excellent reusability. What's more, mag GO–CS–Ti⁴⁺ was applied to capturing phosphopeptides from real biological samples, human serum from both healthy people and patients and nonfat milk. The results successfully manifested that the mag GO–CS–Ti⁴⁺ could be a great affinity material for the enrichment of low-abundant phosphopeptides from biological samples.