Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.
J Chromatogr A. 2011 Aug 12;1218(32):5504-11. doi: 10.1016/j.chroma.2011.06.049. Epub 2011 Jun 21.
The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.
采用聚（苯乙烯-共-二乙烯基苯）整体毛细管柱在高效液相色谱中对完整蛋白质（包括各种氨基酸修饰产生的蛋白质同工型）进行分离，并在线连接飞行时间质谱仪（MS）进行描述。使用 250mm×0.2mm 的整体毛细管柱，在 2h 线性梯度和流动相中添加甲酸作为离子对试剂的情况下，可实现>600 的高峰容量的高灵敏度分离。高分辨率色谱与高精度 MS 的结合能够区分仅在氧化和生物素化状态上有所不同的蛋白质同工型，从而实现结构同工型的分离。最后，讨论了由于糖基化而分离蛋白质同工型的潜力。
