Two-dimensional guanidinium-based covalent organic nanosheets for controllable recognition and specific enrichment of global/multi-phosphopeptides

Highly specific capture of phosphopeptides, especially multi-phosphopeptides, from complex biological samples is critical for comprehensive phosphoproteomic analysis, but it still poses great challenges due to the lack of affinity material with ideal enrichment efficiency. Here, two-dimensional (2D) covalent organic framework (COFs) nanosheets was applied for selective separation of phosphopeptides for the first time. Particularly, by incorporating guanidinium units, the 2D guanidinium-based COF nanosheets (denoted as TpTG(Cl) CONs) exhibited controllable and specific enrichment performance towards global/multi-phosphopeptides. TpTG(Cl) CONs was easy to prepare and showed large surface area, low steric hindrance, abundant accessible interaction sites and high chemical stability. Taking these merits together, TpTG(Cl) CONs exhibited excellent efficiency for phosphopeptide enrichment, such as low detection limits (0.05 fmol mu L-1 for global phosphopeptides and 0.1 fmol mu L-1 for multi-phosphopeptides), high selectivity (1:5000 of molar ratios of beta-casein/BSA for both global and multi-phosphopeptides), high adsorption capacity (100 mg g(-1) for global phosphopeptides and 50 mg g(-1) for multi-phosphopeptides). Furthermore, TpTG(Cl) CONs could be reused due to the high chemical stability. In addition, TpTG(Cl) CONs were successfully applied to controllable and specific capture of endogenous global/multi-phosphopeptides from human serum and human saliva, indicating its good potential in rapid and sensitive detection of biomarkers from biological fluid. Finally, rat liver protein digest was used to confirm the high specificity of TpTG(Cl) CONs towards multi-phosphopeptides and demonstrated its potential as an ideal enrichment probe for comprehensive phosphoproteomic analysis.

Automated summary

In this study, 2D guanidinium-based COF nanosheets were utilized for the selective separation of phosphopeptides with high enrichment efficiency and controllability. The nanosheets showed excellent adsorption capacity and chemical stability towards both global and multi-phosphopeptides.