Department of Chemistry, The University of Hong Kong, Hong Kong, P R China.
Proteomics. 2011 Jun;11(11):2308-19. doi: 10.1002/pmic.201100110. Epub 2011 May 5.
Herein, we describe the development of a fully automatable technology that features online coupling of high-pH RP separation with conventional low-pH RP separation in a two-dimensional capillary liquid chromatography (2-D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first-dimension, high-pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second-dimension, low-pH RP separation, each under identical gradient-elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non-redundant proteins, of which 50% were observed in all three replicates. A comparison of RP-RP 2-D LC and strong cation exchange-RP 2-D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.
在这里，我们描述了一种全自动技术的发展，该技术在二维毛细管液相色谱（2-D LC）系统中具有在线偶联高 pH RP 分离和常规低 pH RP 分离的功能，用于 shotgun 蛋白质组学分析。完整的分析包括 13 个分离周期，每个周期都涉及将第一维洗脱液（高 pH RP 分离）转移到第二 RP 维度进行进一步分离。在 13 个馏分（周期）中，溶剂强度逐渐增加，以洗脱所有肽段，然后在第二维、低 pH RP 分离下进一步分离，每个馏分在相同的梯度洗脱条件下进行。每次分析的总运行时间为 52 小时。在对小鼠胚胎成纤维细胞裂解物的三次重复分析中，我们使用该技术鉴定了 2431 个非冗余蛋白，其中 50%在所有三个重复中都被观察到。RP-RP 2-D LC 和强阳离子交换-RP 2-D LC 分析的比较表明，这两种技术主要识别不同的肽段，从而突出了它们分离化学的差异。
