Synthesis of a metal oxide affinity chromatography magnetic mesoporous nanomaterial and development of a one-step selective phosphopeptide enrichment strategy for analysis of phosphorylated proteins.
Synthesis of a metal oxide affinity chromatography magnetic mesoporous nanomaterial and development of a one-step selective phosphopeptide enrichment strategy for analysis of phosphorylated proteins.
复制标题
DOI：
10.1016/j.aca.2022.339430
复制
发表时间：
2022-01
影响因子：
6.2
通讯作者：
Lu Gao;Jiaqing Tao;Likai Qi;Xinyi Jiang;Hui-Qing Shi;Yahui Liu;Bin Di;Yajing Wang;Fang Yan
中科院分区：
文献类型：
--
作者：
Lu Gao;Jiaqing Tao;Likai Qi;Xinyi Jiang;Hui-Qing Shi;Yahui Liu;Bin Di;Yajing Wang;Fang Yan
蛋白质磷酸化是一种重要的蛋白质翻译后修饰，它调节着蛋白质结构的改变、分子间的相互作用、细胞的增殖和凋亡以及各种恶性疾病等多种生物学过程。尽管基于质谱（MS）的技术提供了一种可行的策略来分析磷酸化蛋白质，但由于复杂的制备和必要的磷酸肽富集的样品损失，生物样品中磷酸肽的无标记定量分析仍然是一项具有挑战性的任务。本研究制备了粒径为500 nm的磁性介孔TiO 2微球Fe3O4@nSiO2@mSiO2@TiO2。合成的材料表现出良好的选择性（β-酪蛋白：BSA的摩尔比= 1：2000）、高灵敏度（0.5 fmol μL−1β-酪蛋白）和良好的吸附容量（120 mg g−1）。此外，富集材料表面的介孔具有独特的尺寸排阻特性，可以“捕获"胰蛋白酶并形成临时固定化酶反应器，使胰蛋白酶消化过程从至少16 h缩短至2 h。在此基础上，成功建立了一种简便的一步选择性磷酸肽富集方法，该方法包括蛋白质消化和磷酸肽富集两个步骤，并将其应用于脱脂牛奶和HepG 2细胞等复杂生物样品中磷酸肽的选择性富集，表明该方法在真实的样品分析中具有很好的可行性和应用潜力。更重要的是，结合质谱技术，建立了一种无标记的磷酸化蛋白质组学定量分析方法，并对含有BSA和不同浓度β-酪蛋白的混合样品进行了测定。结果表明，β-酪蛋白独特磷酸肽的强度（m/z2061.77）与β-酪蛋白的浓度呈线性关系，R = 0.9987。本研究为选择性富集磷酸肽和简便的磷酸化蛋白质的无标记相对定量分析提供了一种新的分析策略。
Protein phosphorylation is one of the most vital protein post-translational modifications, which regulates numerous biological processes including protein structure transformation, molecular interaction, cell proliferation and apoptosis, as well as various malignant diseases. Though mass spectrometry (MS)-based technologies provide a feasible strategy to analyze phosphorylated proteins, label-free quantitative analysis for phosphopeptides in biological samples is still a challenging task due to sample losses from complicated preparation and necessary phosphopeptide enrichment. In this study, magnetic mesoporous TiO2microspheres, Fe3O4@nSiO2@mSiO2@TiO2, a metal oxide affinity chromatography material, were synthesized with uniform particle size of 500 nm. The synthesized material exhibited good selectivity (molar ratio ofβ-casein:BSA = 1:2000, great sensitivity (0.5 fmol μL−1β-casein), and good adsorption capacity (120 mg g−1). Moreover, the proper mesopores on the surface of the enrichment material displayed the unique property of size-exclusion, which could ‘capture’ the trypsin and form a temporary immobilized enzyme reactor, reducing the tryptic digestion procedure from at least 16 h to 2 h. Based on the synthesized material, a facile one-step selective phosphopeptide enrichment method was successfully developed and consisted with protein digestion and phosphopeptide enrichment, which was further applied to selectively enrich phosphopeptides from the complex biological samples of nonfat milk and HepG2 cells, indicating its great feasibility and potential of applications in real sample analysis. More importantly, combining with MS, a label-free phosphoproteomic quantitative strategy was established and performed for determination of the mixture samples containing BSA and various concentrations ofβ-casein. The results showed that the obtained calibration curve was linear with R = 0.9987 by plotting the intensities of unique phosphopeptide ofβ-casein (m/z2061.77) versus the concentrations ofβ-casein. Our study will provide a novel analytical strategy for selective enrichment of phosphopeptides and facile label-free relative quantitative analysis of phosphorylated proteins.