Department of Chemistry, The University of Hong Kong, Hong Kong, China.
J Sep Sci. 2012 Jul;35(14):1755-63. doi: 10.1002/jssc.201200054.
We have developed a fully automatable two-dimensional liquid chromatography platform for shotgun proteomics analyses based on the online coupling of hydrophilic interaction liquid chromatography (HILIC) - using a nonionic type of TSKgel Amide 80 at either pH 6.8 (neutral) or 2.7 (acidic) - with conventional low-pH reversed-phase chromatography. Online coupling of the neutral-pH HILIC and reversed phase chromatography systems outperformed the acidic HILIC-reversed phase chromatography combination, resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172 unique peptides) increases in the number of identified peptides and proteins from duplicate analyses of Rat pheochromocytoma lysates. Armed with this optimized HILIC-reversed phase liquid chromatography platform, we identified 2554 nonredundant proteins from duplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately 41 to 10(6) copies per cell, which contained up to approximately 2092 different validated protein species with a dynamic range of concentrations of up to approximately 10(4) . This present study establishes a fully automated platform as a promising methodology to enable online coupling of different hydrophilic HILIC and reversed phase chromatography systems, thereby expanding the repertoire of multidimensional liquid chromatography for shotgun proteomics.
我们开发了一种完全自动化的二维液相色谱平台，用于基于亲水相互作用液相色谱（HILIC）的蛋白质组学分析，其中在线偶联非离子型 TSKgel Amide 80，分别在 pH 6.8（中性）或 2.7（酸性）与常规低 pH 反相色谱法。中性 pH 值 HILIC 与反相色谱系统的在线偶联优于酸性 HILIC-反相色谱组合，从而使重复分析大鼠嗜铬细胞瘤裂解物时鉴定的肽和蛋白质数量分别增加了 18.4%（1914 对 1617 个非冗余蛋白质）和 41.6%（12989 对 9172 个独特肽）。利用这种优化的 HILIC-反相液相色谱平台，我们从重复分析酵母裂解物的实验中鉴定了 2554 个非冗余蛋白质，检测到的蛋白质丰度约为每个细胞 41 到 10^6 拷贝，其中包含多达约 2092 种不同的已验证蛋白质，浓度范围高达约 10^4。本研究建立了一种完全自动化的平台，作为一种有前途的方法，能够实现不同亲水 HILIC 和反相色谱系统的在线偶联，从而扩展用于蛋白质组学的多维液相色谱方法。
