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 μL ^-1 for global phosphopeptides and 0.1 fmol μL ^-1 for multi-phosphopeptides), high selectivity (1:5000 of molar ratios of β-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.
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