A silica-based immobilized metal affinity chromatography (IMAC) sorbent with the morphological properties suitable for purification of large phosphorylated biomolecules was synthesized. The sorbent was designed in the form of monodisperse-porous silica microspheres, 5.3 μm in size, having bimodal pore size distribution with a large median pore size (40 nm) and high surface area (163 m2/g) decorated with Ti(IV) cations (i.e. Ti(IV)@THSPMP@SiO2 microspheres). The decoration of silica microspheres with Ti(IV) cations was made by using 3-(trihydroxysilyl)propyl methylphosphonate (THSPMP) as a bifunctiontional linker, by preserving their bimodal pore size distribution. The mesopores provided a large surface area for parking of adsorbed phosphoproteins as large phosphorylated biomolecules while the intraparticular transport of phosphoproteins was facilitated by the macropores providing a large median pore size. High equilibrium adsorption capacity and high desorption yield in the purification of phosphoproteins were obtained using Ti(IV)@THSPMP@SiO2 microspheres as the sorbent in batch- and microfluidic-IMAC systems. The phosphoproteins, α-casein and β-casein were isolated from milk and human serum with almost quantitative yields and high purity in the batch IMAC system. The appropriate microcolumn permeability (3.66 × 10-14 m2) originating from its appropriate average diameter (5.3 μm), high porosity (0.948 cm3/g) and high surface area (163 m2/g) of Ti(IV)@THSPMP@SiO2 microspheres makes the synthesized sorbent a promising stationary phase for dynamic chromatography. Hence, a new phosphoprotein enrichment format, a microfluidic IMAC system was constructed and successfully operated for highly selective purification of phosphoproteins from non-fat milk as a complex sample. The microfluidic-IMAC system is a promising tool particularly for phosphoproteomic applications performed using samples in microliter or nanoliter scale, also involving an on-line connection of purification unit to LC-MS for the identification of large phosphorylated biomolecules enriched.

中文翻译：

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基于Ti（IV）修饰的二氧化硅微球的微流固定金属亲和色谱，用于纯化磷蛋白。

合成了具有适合纯化大型磷酸化生物分子的形态学特性的二氧化硅基固定化金属亲和色谱（IMAC）吸附剂。吸附剂以单分散多孔二氧化硅微球的形式设计，尺寸为5.3μm，具有双峰孔径分布，中值孔径（40 nm）大，表面积高（163 m2 / g）用Ti（IV）装饰阳离子（即Ti（IV）@ THSPMP @ SiO2微球）。通过使用3-（三羟基甲硅烷基）丙基甲基膦酸酯（THSPMP）作为双功能接头，通过保留其双峰孔径分布，对带有Ti（IV）阳离子的二氧化硅微球进行了修饰。中孔为大分子磷酸化的生物分子提供了大的表面积，用于吸附吸附的磷蛋白，而磷蛋白的颗粒内运输则由大孔提供了较大的中值孔径。以Ti（IV）@ THSPMP @ SiO2微球为吸附剂在分批和微流IMAC系统中获得了高平衡吸附能力和高脱附产率的磷蛋白。从批料IMAC系统中以几乎定量的产量和高纯度从牛奶和人血清中分离了磷蛋白，α-酪蛋白和β-酪蛋白。适当的微柱渗透率（3.66×10-14 m2）源自其适当的平均直径（5.3μm），高孔隙率（0。Ti（IV）@ THSPMP @ SiO2微球的948 cm3 / g和高表面积（163 m2 / g）使得合成的吸附剂成为动态色谱的有希望的固定相。因此，构建了一种新的磷蛋白富集形式，即微流IMAC系统，并成功运行，可从非脂奶中以复杂样品的高选择性纯化磷蛋白。微流体-IMAC系统是一个有前途的工具，特别是对于使用微升或纳升规模的样品进行的磷酸蛋白质组学应用，还涉及将纯化单元与LC-MS在线连接，以鉴定富集的大型磷酸化生物分子。构建了微流体IMAC系统并成功运行，可从脱脂牛奶中以复杂样品的高选择性纯化磷蛋白。微流体-IMAC系统是一个有前途的工具，特别是对于使用微升或纳升规模的样品进行的磷酸蛋白质组学应用，还涉及将纯化单元与LC-MS在线连接，以鉴定富集的大型磷酸化生物分子。构建了微流体IMAC系统并成功运行，可从脱脂牛奶中以复杂样品的高选择性纯化磷蛋白。微流体-IMAC系统是一个有前途的工具，特别是对于使用微升或纳升规模的样品进行的磷酸蛋白质组学应用，还涉及将纯化单元与LC-MS在线连接，以鉴定富集的大型磷酸化生物分子。