Quantitative Analysis of Proteome Coverage and Recovery Rates for Upstream Fractionation Methods in Proteomics
Quantitative Analysis of Proteome Coverage and Recovery Rates for Upstream Fractionation Methods in Proteomics
复制标题
DOI：
10.1021/pr901063t
复制
发表时间：
2010-04-01
影响因子：
4.4
通讯作者：
Foster, Leonard J.
中科院分区：
文献类型：
--
作者：
Fang, Yuan;Robinson, Dale P.;Foster, Leonard J.
任何细胞甚至任何亚细胞部分的蛋白质组都过于复杂，无法通过一维液相色谱-串联质谱（LC-MS/MS）进行完整分析。因此，为了对感兴趣的蛋白质组进行更深入的覆盖，大多数小组在LC-MS/MS之前常规地对样品进行减法处理，以便进入LC-MS/MS的材料比原始样品更简单。生物化学家已经使用了几十年的蛋白质和/或肽的分离方法，如强阳离子交换色谱（SCX）、等电聚焦（IEF）和SDS-PAGE，是目前最常用的预分离方法。到目前为止，还没有对各种方法的相对优点进行全面的、有控制的评价，尽管已经进行了一些二元比较。在这里，我们比较了蛋白质和肽水平上最流行的样品分离方法，复制所有分析以提供分析变异性的估计，并精确控制专用于每次分析的仪器时间，以及直接测量从每个分离程序中提取的蛋白质或肽的回收率。对于最大的蛋白质组覆盖率，SDS-PAGE显然是最有效的测试方法，超过90%的整个数据集被发现。当考虑到每次分馏过程后回收的物料量时，基于溶液的IEF和SCX的表现相似，大约80%的输入被回收。
The proteome of any cell or even any subcellular fraction remains too complex for complete analysis by one dimension of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hence, to achieve greater depth of coverage for a proteome of interest, most groups routinely subfractionate the sample prior to LC-MS/MS so that the material entering LC-MS/MS is less complex than the original sample. Protein and/or peptide fractionation methods that biochemists have used for decades, such as strong cation exchange chromatography (SCX), isoelectric focusing (IEF) and SDS-PAGE, are the most common prefractionation methods used currently. There has, as yet, been no comprehensive, controlled evaluation of the relative merits of the various methods, although some binary comparisons have been made. Here, we compare the most popular methods for fractionating samples at both the protein and peptide level, replicating all analyses to provide estimates of the variability in the analyses and controlling precisely for instrument time dedicated to each analysis, as well as directly measuring the recovery of protein or peptide from each fractionation procedure. For maximal proteome coverage, SDS-PAGE is very clearly the most effective method tested, with more than 90% of the entire data set found. When considering the amount of material recovered after each fractionation procedure, solution-based IEF and SCX performed similarly, with approximately 80% of the input being recovered.