Department of Chemistry, University of Oslo, Blindern, Oslo, Norway.
J Sep Sci. 2013 Sep;36(17):2838-47. doi: 10.1002/jssc.201300499.
The single-run resolving power of current 10 μm id porous-layer open-tubular (PLOT) columns has been optimized. The columns studied had a poly(styrene-co-divinylbenzene) porous layer (~0.75 μm thickness). In contrast to many previous studies that have employed complex plumbing or compromising set-ups, SPE-PLOT-LC-MS was assembled without the use of additional hardware/noncommercial parts, additional valves or sample splitting. A comprehensive study of various flow rates, gradient times, and column length combinations was undertaken. Maximum resolution for <400 bar was achieved using a 40 nL/min flow rate, a 400 min gradient and an 8 m long column. We obtained a 2.3-fold increase in peak capacity compared to previous PLOT studies (950 versus previously obtained 400, when using peak width = 2σ definition). Our system also meets or surpasses peak capacities obtained in recent reports using nano-ultra-performance LC conditions or long silica monolith nanocolumns. Nearly 500 proteins (1958 peptides) could be identified in just one single injection of an extract corresponding to 1000 BxPC3 beta catenin (-/-) cells, and ~1200 and 2500 proteins in extracts of 10,000 and 100,000 cells, respectively, allowing detection of central members and regulators of the Wnt signaling pathway.
当前 10 μm id 多孔层开管（PLOT）柱的单运行分辨率已得到优化。研究的柱子具有聚（苯乙烯-共-二乙烯基苯）多孔层（~0.75 μm 厚）。与许多以前采用复杂管道或妥协设置的研究不同，SPE-PLOT-LC-MS 的组装未使用额外的硬件/非商业部件、额外的阀门或样品分流。对各种流速、梯度时间和柱长组合进行了全面研究。在<400 bar 下，使用 40 nL/min 的流速、400 min 的梯度和 8 m 长的柱子可实现最大分辨率。与以前的 PLOT 研究相比，我们的系统获得了 2.3 倍的峰容量增加（950 比以前使用峰宽=2σ 定义时获得的 400）。我们的系统还满足或超过了最近使用纳超高效 LC 条件或长硅胶整体纳米柱获得的峰容量。仅在 1000 BxPC3 β-连环蛋白（-/-）细胞提取物的一次单注射中，就可以鉴定近 500 种蛋白质（1958 种肽），而在 10,000 个和 100,000 个细胞提取物中，分别可以鉴定出约 1200 种和 2500 种蛋白质，从而可以检测到 Wnt 信号通路的中心成员和调节剂。
