US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA.
J Sep Sci. 2012 Jun;35(12):1399-405. doi: 10.1002/jssc.201100865.
In proteomic investigations, a number of different separation techniques can be applied to fractionate whole cell proteomes into more manageable fractions for subsequent analysis. In this work, utilizing HPLC and mass spectrometry for protein identification, two different fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a bacterial proteome. Column-based chromatofocusing and liquid-based isoelectric focusing both utilized pH gradients to produce similar results in terms of the numbers of proteins successfully identified (402 and 378 proteins) and the consistency of proteins identified from one experiment to the next (<10% change). However, there was limited overlap in the protein sets with <50% of the proteins identified as common between the sets of proteins identified by the different systems. In addition to the numbers of proteins identified and consistency of those identified, the reduced monetary costs of experimentation and increased assay flexibility produced by using isoelectric focusing was considered in order to adopt a system best suited for comparative proteomic projects.
在蛋白质组学研究中，可以应用许多不同的分离技术将整个细胞蛋白质组分离成更易于管理的部分，以便随后进行分析。在这项工作中，我们利用 HPLC 和质谱法进行蛋白质鉴定，比较和对比了两种不同的分馏方法，以确定每种方法在简单和可重复地分离细菌蛋白质组方面的潜力。基于柱的色谱聚焦和基于液体的等电聚焦都利用 pH 梯度产生了相似的结果，就成功鉴定的蛋白质数量（402 种和 378 种）和从一次实验到下一次实验鉴定的蛋白质的一致性（<10%的变化）而言。然而，蛋白质组之间的重叠有限，<50%的蛋白质被鉴定为不同系统鉴定的蛋白质组之间的共有蛋白质。除了鉴定的蛋白质数量和鉴定的蛋白质的一致性外，还考虑了使用等电聚焦产生的实验成本降低和测定灵活性增加，以便采用最适合比较蛋白质组学项目的系统。
