Institut Galien Paris Sud, UMR 8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Univ. Paris-Saclay, 5 rue Jean Baptiste Clément, 92290 Châtenay-Malabry, France.
Univ. Paris-Est, ICMPE (UMR7182), CNRS, UPEC, Thiais, 94320, France.
Analyst. 2017 Jan 26;142(3):485-494. doi: 10.1039/c6an02324j.
A microdevice combining online preconcentration and separation of phosphopeptides was developed in a glass microchip. An ethylene glycol methacrylate phosphate (EGMP), acrylamide (AM) and bisacrylamide (BAA) based monolith was synthesized within microchannels through a photo-driven process. Morphological investigations revealed a homogeneous monolithic structure composed of uniform nodules (∼0.8 μm), with a large pore volume (0.62 cm g) and sufficiently high specific surface area (34.1 m g). These features make the monolith particularly interesting for preconcentration purposes. Immobilization of Zr ions on the phosphate groups present at the poly(EGMP-co-AM-co-BAA) monolith surface leads to immobilized metal affinity chromatography support. This monolith-Zr showed a great capacity to capture phosphopeptides. Successful preconcentration and separation of a mixture of ERK2 derived peptides differing only by their phosphorylation degree and sites could be achieved with signal enhancement factors between 340 and 910 after only 7 min of preconcentration. This integrated microdevice represents a novel approach for phosphoproteomic applications.
一种在线浓缩和分离磷酸肽的微器件在玻璃微芯片中得以开发。通过光驱动过程，在微通道内合成了一种基于乙二醇甲基丙烯酸磷酸酯（EGMP）、丙烯酰胺（AM）和双丙烯酰胺（BAA）的整体式柱。形貌研究表明，整体式柱由均匀的小珠（约 0.8μm）组成，具有较大的孔体积（0.62cm3/g）和足够高的比表面积（34.1m2/g）。这些特点使得整体式柱特别适合于浓缩目的。Zr 离子固定在聚（EGMP-co-AM-co-BAA）整体式柱表面的磷酸基团上，形成固定金属亲和色谱支撑物。这种整体柱-Zr 对磷酸肽具有很强的捕获能力。在仅 7 分钟的浓缩时间后，通过信号增强因子在 340 到 910 之间，成功地对仅在磷酸化程度和位点上有所不同的 ERK2 衍生肽混合物进行了预浓缩和分离。这种集成的微器件为磷酸蛋白质组学应用提供了一种新方法。
