Wang Fangjun, Dong Jing, Ye Mingliang, Wu Ren'an, Zou Hanfa
Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
J Chromatogr A. 2009 May 1;1216(18):3887-94. doi: 10.1016/j.chroma.2009.02.082. Epub 2009 Mar 5.
Monolithic columns are widely used in shotgun proteome analysis. However, it is difficult to increase the separation capability and proteome coverage by using conventionally organic polymer-based monolithic column due to the difficulty of controlling homogeneity of the overall pore structure (both pores and microglobules), which leads to relatively low column efficiency. Therefore, we studied the effect of constitute and percentage of porogenic solvent, functional monomer, column length, and separation gradient on the peak capacity and proteome coverage by methacrylate-based reversed phase monolithic columns. It was demonstrated that the porous property of the hydrophobic monolith, which was mainly determined by the porogenic solvent, was crucial to the proteome coverage when similar methacrylate monomer was utilized and a ternary porogenic solvent was adopted to prepare C12 monolithic column with relatively homogeneous overall pore structure. It was also shown that high proteome coverage could be reliably obtained with online multidimensional separation using totally monolithic columns system with the length of analytical column at 85 cm and reversed phase separation gradient at 210 min.
整体柱在鸟枪法蛋白质组分析中被广泛应用。然而，使用传统的基于有机聚合物的整体柱难以提高分离能力和蛋白质组覆盖率，因为难以控制整体孔结构（包括孔和微球）的均匀性，这导致柱效相对较低。因此，我们研究了致孔剂、功能单体、柱长和分离梯度的组成及百分比对基于甲基丙烯酸酯的反相整体柱的峰容量和蛋白质组覆盖率的影响。结果表明，当使用相似的甲基丙烯酸酯单体并采用三元致孔剂制备具有相对均匀整体孔结构的C12整体柱时，疏水性整体柱的多孔性质（主要由致孔剂决定）对蛋白质组覆盖率至关重要。研究还表明，使用分析柱长度为85 cm且反相分离梯度为210 min的全整体柱系统进行在线多维分离，可以可靠地获得高蛋白质组覆盖率。
