Ning Zhi-Bin, Li Qing-Run, Dai Jie, Li Rong-Xia, Shieh Chia-Hui, Zeng Rong
Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai 200031, China.
J Proteome Res. 2008 Oct;7(10):4525-37. doi: 10.1021/pr800318j. Epub 2008 Sep 11.
The complexity and diversity of biological samples in proteomics require intensive fractionation ahead of mass spectrometry identification. This work developed a chromatographic method called virtual three-dimensional chromatography to fractionate complex protein mixtures. By alternate elution with different pHs and salt concentrations, we implemented pH and salt steps by turns on a single strong cation exchange column to fully exploit its chromatographic ability. Given standard proteins that were not resolved solely by pH or salt gradient elution could be successfully separated using this combined mode. With a reversed phase column tandem connected behind, we further fractionated as well as desalted proteins as the third dimension. This present strategy could readily be adapted with respect to special complexity of biological samples. Crude plasma without depleting high abundance proteins were fractionated by this three-dimensional mode and then analyzed by reversed phase liquid chromatography coupled with LTQ mass spectrometry. In total, 1933 protein groups with wide dynamic ranges were identified from a single experiment. Some characteristics that correlated to the behavior of proteins on strong cation exchange columns are also discussed.
蛋白质组学中生物样品的复杂性和多样性要求在质谱鉴定之前进行密集分级分离。这项工作开发了一种称为虚拟三维色谱的色谱方法来分离复杂的蛋白质混合物。通过用不同pH值和盐浓度交替洗脱，我们在单个强阳离子交换柱上依次实现pH和盐梯度，以充分发挥其色谱能力。结果表明，仅通过pH或盐梯度洗脱无法分离的标准蛋白质可以使用这种组合模式成功分离。在后面串联连接一个反相柱，我们进一步对蛋白质进行分级分离和脱盐，作为第三维。这种目前的策略可以很容易地根据生物样品的特殊复杂性进行调整。通过这种三维模式对未去除高丰度蛋白质的粗血浆进行分级分离，然后通过反相液相色谱结合LTQ质谱进行分析。单次实验共鉴定出1933个具有宽动态范围的蛋白质组。还讨论了一些与蛋白质在强阳离子交换柱上行为相关的特性。
