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

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.