Univ Paris-Sud, Laboratoire des Protéines et Nanotechnologies en Sciences Séparatives, Faculté de Pharmacie, Châtenay-Malabry, France.
J Sep Sci. 2010 Apr;33(8):1090-8. doi: 10.1002/jssc.200900713.
We evaluated the potential of CE to analyse different isoforms of unphosphorylated recombinant tau protein and for separating one phosphorylated tau from the respective unphosphorylated protein. Different capillary coatings such as polyacrylamide, poly-(ethylene oxide) and polybrene (PB) were evaluated to overcome the poor efficiencies obtained with fused-silica capillary. Although peak asymmetry values were quite similar for the three investigated coatings, the peak efficiencies were 35-fold and 5-fold higher with PB coating than with polyacrylamide and poly(ethylene oxide) coatings, respectively. The recovery percentage (over 97%) was satisfactory and confirmed the efficacy of PB coating to limit the adsorption of tau protein to capillary walls. Moreover, PB coating produced higher repeatability for migration times (RSD values <1.2%) in comparison to the neutral coatings. The potential of PB-modified capillary in producing high resolutive separations of one phosphorylated tau isoform from its unphosphorylated counterpart and of a mixture of phosphorylated and unphosphorylated tau peptides was demonstrated with 50 mM phosphate buffer pH 3.0. The separation of unphosphorylated tau isoform 352 (Tau-352) from Tau-352 phosphorylated in vitro by the mitogen-activated protein kinase ERK2, was accomplished in less than 15 min.
我们评估了 CE 分析不同未磷酸化重组 tau 蛋白异构体的潜力，并用于分离磷酸化 tau 与相应的未磷酸化蛋白。为了克服熔融硅毛细管获得的低效率，我们评估了不同的毛细管涂层，如聚丙烯酰胺、聚（环氧乙烷）和多聚（亚胺）（PB）。尽管三种研究涂层的峰不对称值非常相似，但 PB 涂层的峰效率分别比聚丙烯酰胺和聚（环氧乙烷）涂层高 35 倍和 5 倍。回收率（超过 97%）令人满意，证实了 PB 涂层限制 tau 蛋白吸附到毛细管壁的效果。此外，与中性涂层相比，PB 涂层产生的迁移时间重复性更高（RSD 值 <1.2%）。使用 50 mM 磷酸盐缓冲液 pH 3.0 证明了 PB 修饰毛细管在产生高分辨率分离一个磷酸化 tau 异构体与其未磷酸化对应物以及磷酸化和未磷酸化 tau 肽混合物方面的潜力。通过丝裂原活化蛋白激酶 ERK2 在体外对 Tau-352 进行磷酸化，成功地在不到 15 分钟内分离出未磷酸化的 Tau-352 异构体。
