Design of guanidyl-functionalized magnetic covalent organic framework for highly selective capture of endogenous phosphopeptides.

The identification and analysis of endogenous phosphopeptides is still a scientific challenge due to their low abundance and the complicated matrix of bio-samples. Hence, the highly effective enrichment of endogenous phosphopeptides is a prerequisite for comprehensive phosphoproteome analysis. Here, a novel guanidyl-functionalized magnetic covalent organic framework (denoted as SPIO@COF-Guanidyl) nanosphere was designed for selective and efficient phosphopeptide enrichment. The SPIO@COF-Guanidyl nanospheres possessed specific recognition sites of functional guanidyl groups, large surface area, regular mesoporous structure, and superior magnetic responsiveness, resulting in excellent performances in phosphopeptide enrichment with high selectivity (β-casein/BSA = 1:1000), extremely high sensitivity (5 × 10 M), an excellent size-exclusion effect (β-casein digests/β-casein/bovine serum albumin (BSA) = 1:500:500), and good reusability (at least 5 times). In addition, due to the synergistic effect of functional molecules and the size-selection effect of the COF structure, SPIO@COF-Guanidyl nanospheres captured 63 unique endogenous phosphopeptides from a 10 μL sample of human saliva. Therefore, SPIO@COF-Guanidyl nanospheres possess a huge potential for trace biological detection and endogenous phosphopeptide analysis.