Kim Hyeyeung, Lubman David M
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
J Chromatogr A. 2008 Jun 13;1194(1):3-10. doi: 10.1016/j.chroma.2008.03.065. Epub 2008 Mar 27.
Multi-dimensional liquid-based separation is required for fractionation and mapping of complex protein mixtures from cells. A method that has been used as the first dimension in such separations is chromatofocusing (CF), which is based on generating a pH gradient on an anion exchange column. The use of pH in the first dimension is essential where pH is a fundamental property of proteins and can provide information on post-translationally modified forms of a protein. In this work, a micro-chromatofocusing technique is introduced which can separate microgram levels of proteins from cell lysates for further analysis by LC-MS/MS. It is shown that this method can analyze 10 microg of sample and detect nearly 700-800 proteins from ovarian cancer cell line lysates.
对细胞中复杂蛋白质混合物进行分级分离和图谱分析需要多维液相分离。在这种分离中用作第一维的一种方法是色谱聚焦（CF），它基于在阴离子交换柱上产生pH梯度。在第一维中使用pH至关重要，因为pH是蛋白质的基本属性，并且可以提供有关蛋白质翻译后修饰形式的信息。在这项工作中，引入了一种微色谱聚焦技术，该技术可以从细胞裂解物中分离出微克级的蛋白质，以便通过LC-MS/MS进行进一步分析。结果表明，该方法可以分析10微克样品，并从卵巢癌细胞系裂解物中检测到近700-800种蛋白质。
