Phosphorylation is one of the most important post-translational modifications of proteins, but due to the low abundance of phosphopeptides, enrichment is an essential step before mass spectrometric analysis. Although there are a number of enrichment methods developed targeting different forms of proteins phosphorylations, there are few reports on specific recognition and capture of single phosphopeptide. Herein, based on the advantages of dual affinity of TiO₂ and urea to a phosphate group and molecular imprinting towards the peptide sequence, the precise recognition of intact phosphorylated peptides was successfully achieved. The same peptide sequence with different phosphorylation forms (c.a. Ser, Thr and Tyr) were used as templates for proof-of-principle study, and the imprinted particles were successfully synthesized, characterized, and have the capacity to specifically recognize the targeted unique phosphorylation excluding even its isoforms. In addition, the produced molecularly imprinted nanoparticles have numerous important advantages, including strong affinity, high specificity toward single phosphopeptides, tolerance to interferences, fast binding kinetics, substantial binding capacity, excellent stability and reusability, making them an ideal sorbent for specific enrichment of unique phosphopeptides. Finally, different phosphorylation forms were specifically enriched from both standard peptides’ mixture and casein/milk digests.

磷酸化是蛋白质最重要的翻译后修饰之一，但由于磷酸化肽的丰度较低，因此富集是质谱分析前必不可少的步骤。尽管针对不同形式的蛋白质磷酸化开发了许多富集方法，但关于特异性识别和捕获单个磷酸肽的报道很少。在此，基于TiO _{2的双重亲和性的优点}和脲对磷酸基团和对肽序列的分子印迹，成功地实现了完整磷酸化肽的精确识别。以具有不同磷酸化形式（ca Ser、Thr和Tyr）的相同肽序列作为模板进行原理验证研究，成功合成、表征了印迹颗粒，并具有特异性识别靶向独特磷酸化的能力，不包括甚至它的异构体。此外，所制备的分子印迹纳米粒子具有许多重要的优点，包括亲和力强、对单个磷酸肽的特异性高、抗干扰性强、结合动力学快、结合能力强、稳定性和可重复使用性好、使其成为特异性富集独特磷酸肽的理想吸附剂。最后，从标准肽混合物和酪蛋白/牛奶消化物中特异性富集了不同的磷酸化形式。