Gao Mingxia, Zhang Jie, Deng Chunhui, Yang Pengyuan, Zhang Xiangmin
Department of Chemistry and Research Center of Proteome, Fudan University, Shanghai 200433, China.
J Proteome Res. 2006 Oct;5(10):2853-60. doi: 10.1021/pr0602186.
In this study, for the first time, a comprehensive two-dimensional (2D) liquid-phase separation system, coupling strong cation exchange chromatography (SCX) to reversed-phase high performance liquid chromatography (RPLC), instead of specificity depletion method, was developed at the intact protein level for depletion of high-abundance proteins from rat liver. Proteins were prefractionated by SCX in the first dimensional separation, followed by RPLC with high resolution separation. UV absorption intensity was used to differentiate high-abundance proteins. The proteins with the absorbance intensity above 0.1 AU were defined as high abundance proteins and depleted. After removal of high-abundance proteins; other proteins were pooled, digested, and subsequently separated by capillary liquid chromatography coupled with MALDI-TOF/TOF mass spectrometry analysis. The high efficiency of the strategy was demonstrated by analyzing the soluble protein extracted from rat liver tissue. In total, 77 high-abundance proteins were depleted in one experiment flow. The ratio of depleted content of high-abundance proteins to that of total proteins was about 34.5%. In total, 1530 proteins were identified using the depletion strategy. Quantitative estimation of high-abundance proteins through liquid chromatography combined with UV absorption spectra was achieved. On the basis of the reproducible experimental results, a rapid and high-throughput depletion protocol was put forward. Along with depletion of the most (79.1%) high-abundance proteins and the separation of digested peptides, the total separation time could be less than 30 h. This strategy has no bias for depleting high-abundance proteins and enhances the number of identified proteins; therefore, it can be widely used in the global proteins analysis.
在本研究中，首次开发了一种完整蛋白质水平的综合二维（2D）液相分离系统，该系统将强阳离子交换色谱（SCX）与反相高效液相色谱（RPLC）联用，而非特异性去除方法，用于从大鼠肝脏中去除高丰度蛋白质。在第一维分离中，蛋白质通过SCX进行预分离，随后通过RPLC进行高分辨率分离。利用紫外吸收强度来区分高丰度蛋白质。吸光度强度高于0.1 AU的蛋白质被定义为高丰度蛋白质并被去除。去除高丰度蛋白质后，将其他蛋白质合并、消化，随后通过毛细管液相色谱结合基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF/TOF）分析进行分离。通过分析从大鼠肝脏组织中提取的可溶性蛋白质，证明了该策略的高效性。在一个实验流程中，总共去除了77种高丰度蛋白质。高丰度蛋白质的去除量与总蛋白量的比值约为34.5%。使用该去除策略共鉴定出1530种蛋白质。实现了通过液相色谱结合紫外吸收光谱对高丰度蛋白质进行定量估计。基于可重复的实验结果，提出了一种快速且高通量的去除方案。在去除大部分（79.1%）高丰度蛋白质并分离消化后的肽段的同时，总分离时间可少于30小时。该策略在去除高丰度蛋白质方面无偏差，且增加了鉴定出的蛋白质数量；因此，它可广泛应用于全局蛋白质分析。
