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首页> 外文期刊>Journal of chromatography, A: Including electrophoresis and other separation methods >Development of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approach
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Development of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approach

机译：shot枪蛋白质组学在线高/低pH反相反相二维液相色谱的开发：反相-强阳离子交换-反相方法

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摘要

Previously, we described an online high-/low-pH RP-RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP-RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP-RP separation. Relative to the RP-RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample.
机译：以前，我们描述了一种在线高/低pH RP-RP LC系统，该系统显示出与SCX-RP相当的高通量，可自动化性和性能。在此，我们报告了RP-RP平台的一种变体RP-SCX-RP，在两个LC尺寸之间有一个额外的SCX捕集柱。与第二维RP结合使用的SCX色谱柱可用作SCX-RP双相色谱柱，用于将肽捕获在来自第一RP色谱柱的洗脱液中。我们通过蛋白质组学分析拟南芥叶绿体样品和小鼠胚胎小鼠成纤维细胞STO细胞裂解物的低微克水平评估了新平台的性能。通常，RP-SCX-RP通过允许检测大量亲水肽来增强蛋白质鉴定。此外，该平台可用于以低微克水平对iTRAQ应用的粗制叶绿体样品进行定量分析。另外，它允许在线除去iTRAQ反应中过量使用的十二烷基硫酸钠和其他化学物质，从而避免了在进行RP-RP分离之前进行耗时的离线SCX净化的需要。相对于RP-RP系统，我们新开发的RP-SCX-RP平台可以检测iTRAQ标记的粗叶绿体蛋白质样品中大量差异表达的蛋白质。

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