Bensaddek Dalila, Nicolas Armel, Lamond Angus I
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
Int J Mass Spectrom. 2015 Nov 30;391:105-114. doi: 10.1016/j.ijms.2015.07.029.
Despite many recent advances in instrumentation, the sheer complexity of biological samples remains a major challenge in large-scale proteomics experiments, reflecting both the large number of protein isoforms and the wide dynamic range of their expression levels. However, while the dynamic range of expression levels for different components of the proteome is estimated to be ∼10, the equivalent dynamic range of LC-MS is currently limited to ∼10. Sample pre-fractionation has therefore become routinely used in large-scale proteomics to reduce sample complexity during MS analysis and thus alleviate the problem of ion suppression and undersampling. There is currently a wide range of chromatographic techniques that can be applied as a first dimension separation. Here, we systematically evaluated the use of hydrophilic interaction liquid chromatography (HILIC), in comparison with hSAX, as a first dimension for peptide fractionation in a bottom-up proteomics workflow. The data indicate that in addition to its role as a useful pre-enrichment method for PTM analysis, HILIC can provide a robust, orthogonal and high-resolution method for increasing the depth of proteome coverage in large-scale proteomics experiments. The data also indicate that the choice of using either HILIC, hSAX, or other methods, is best made taking into account the specific types of biological analyses being performed.
尽管近年来仪器设备有了许多进步，但生物样品的极端复杂性仍是大规模蛋白质组学实验中的一项重大挑战，这既反映在蛋白质异构体数量众多，也体现在其表达水平的广泛动态范围上。然而，虽然蛋白质组不同组分的表达水平动态范围估计约为10⁶，但液相色谱 - 质谱联用（LC-MS）目前的等效动态范围仅限于约10³。因此，样品预分级已在大规模蛋白质组学中常规使用，以降低质谱分析期间的样品复杂性，从而缓解离子抑制和采样不足的问题。目前有多种色谱技术可作为第一维分离方法应用。在此，我们系统地评估了亲水作用液相色谱（HILIC）与强阴离子交换色谱（hSAX）相比，作为自下而上蛋白质组学工作流程中肽段分级第一维的用途。数据表明，除了作为一种用于翻译后修饰（PTM）分析的有用预富集方法外，HILIC还可为大规模蛋白质组学实验中增加蛋白质组覆盖深度提供一种强大、正交且高分辨率的方法。数据还表明，使用HILIC、hSAX或其他方法的选择，最好根据所进行的特定类型生物分析来做出。
