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 × 10⁻¹¹ 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.

内源性磷酸肽的鉴定和分析由于其丰度低和生物样品基质复杂而仍然是一项科学挑战。因此，高效富集内源性磷酸肽是进行全面磷酸化蛋白质组学分析的前提。在这里，一个新颖的胍基官能化的磁性共价有机骨架（称为SPIO @ COF-胍基）纳米球被设计用于选择性和有效的磷酸肽富集。SPIO @ COF-胍基纳米球具有功能性胍基的特异性识别位点，较大的表面积，规则的介孔结构和出色的磁响应性，因此在磷肽富集方面表现出色，选择性高（β-酪蛋白/ BSA = 1：1000） ，极高的灵敏度（5×10 ^-11M），出色的尺寸排阻作用（β-酪蛋白消化物/β-酪蛋白/牛血清白蛋白（BSA）= 1：500：500）和良好的可重用性（至少5次）。此外，由于功能分子的协同作用和COF结构的尺寸选择作用，SPIO @ COF-胍基纳米球从10μL人唾液样品中捕获了63种独特的内源性磷酸肽。因此，SPIO @ COF-胍基纳米球具有微量生物检测和内源性磷酸肽分析的巨大潜力。