Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method
Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method
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DOI：
10.1002/rcm.227
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发表时间：
2001-01-01
影响因子：
2
通讯作者：
Miller, FR
中科院分区：
文献类型：
--
作者：
Chong, BE;Yan, F;Miller, FR
介绍了一种新的二维双柱液相色谱/质谱联用(LC/MS)技术，将聚焦(CF)与无孔反相(NPS-RP)高效液相色谱联用，从人乳腺上皮全细胞裂解液中分离蛋白质。采用电喷雾电离正交加速飞行时间质谱仪(ESI-TOFMS)对NPS-RP-HPLC液相级分进行直接在线分析。这项技术的一个关键优点是，蛋白质可以根据其在第一层析分离维度中的等电点(PI)在液相中从CF柱上被“剥离”。NPS-RP-HPLC柱根据蛋白质的疏水性进一步分离这些pi集中的组分作为第二层析维度。第三个维度涉及使用ESI-TOFMS在线测定分子量。因此，这种方法具有完全自动化的潜力。此外，还生成了等电点与分子量关系的二维蛋白质图，这类似于二维凝胶图像。因此，这项技术可能为研究全细胞裂解产物中蛋白质的差异表达提供了一种手段。版权所有(C)2001 John Wiley&Sons，Ltd.
A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins fan be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pi-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates. Copyright (C) 2001 John Wiley & Sons, Ltd.