Hydrophilic Porous Magnetic Graphene Oxide/Chitosan Composites for the Selective Separation and Enrichment of N-Glyco- and Phosphopeptides

Highly selective enrichment of low abundance N -glyco- and phosphopeptides from intricate biological samples before mass spectrometry detection is a vital prerequisite for in-depth proteomics. A hydrophilic porous magnetic graphene oxide/chitosan composite (mGO/CS beads) was developed and applied to selectively enrich N -glyco- and phosphopeptides in biological samples. The obtained mGO/CS beads showed strong hydrophilicity, large pore structure, and magnetic property. Based on the multivalent hydrophilic interactions between glycan group on N -glycopeptides, phosphate on phosphopeptides, and amino and hydroxyl groups on chitosan, N -glyco- and phosphopeptides could be simultaneously captured by mGO/CS beads and sequentially released by different eluents. The mGO/CS beads exhibited high sensitivity (limit of detection: 1.0 fmol μL –1 for N -glycopeptides and 0.5 fmol μL –1 for phosphopeptides) and high selectivity (IgG/BSA and β-casein/BSA tryptic digests with a mass ratio of 1:100 and 1:200). After using mGO/CS beads treatment, a total of 83 N -glycopeptides assigned to 80 N -glycoproteins and 10 phosphopeptides were identified by mass spectrometry from human serum and nonfat milk, respectively. These experimental results indicated that mGO/CS beads have great potential for highly efficient enrichment of low-abundance N -glyco- and phosphopeptides in complex biological samples.