Evaluating the use of HILIC in large-scale, multi dimensional proteomics: Horses for courses?
Evaluating the use of HILIC in large-scale, multi dimensional proteomics: Horses for courses?
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DOI：
10.1016/j.ijms.2015.07.029
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发表时间：
2015-11-30
影响因子：
1.8
通讯作者：
Lamond AI
中科院分区：
文献类型：
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作者：
Bensaddek D;Nicolas A;Lamond AI
亲水作用液相色谱（HILIC）为二维液相色谱深度蛋白质组学工作流程提供了一种稳健且可重现的离线液相色谱方法。
HILIC与强阴离子交换（hSAX）分级分离部分正交。
基于HILIC的工作流程从9000多种蛋白质中鉴定出约81000种肽段，平均序列覆盖率约为26%。
HILIC非常适合用于亲水性翻译后修饰（PTMs）的研究。
尽管仪器设备近期有了很多进步，但生物样本的高度复杂性仍然是大规模蛋白质组学实验中的一个主要挑战，这既反映了蛋白质异构体数量众多，也反映了它们表达水平的动态范围很宽。然而，虽然蛋白质组不同成分的表达水平动态范围估计约为10⁷ - 10⁸，但液相色谱 - 质谱（LC - MS）的等效动态范围目前仅限于约10⁶。因此，样品预分级分离已在大规模蛋白质组学中常规使用，以降低质谱分析过程中的样品复杂性，从而缓解离子抑制和欠采样问题。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（hSAX）相比，作为自下而上蛋白质组学工作流程中肽段分级分离的第一维的应用情况。数据表明，除了作为一种用于翻译后修饰分析的有用的预富集方法外，HILIC还能为增加大规模蛋白质组学实验中蛋白质组覆盖深度提供一种稳健、正交且高分辨率的方法。数据还表明，选择使用HILIC、hSAX还是其他方法，最好考虑正在进行的特定类型的生物分析。
HILIC provides a robust and reproducible off-line LC method for 2D-LC deep proteomics workflows. HILIC is part-orthogonal to hSAX fractionation. HILIC-based workflow identified ∼81,000 peptides from >9000 proteins with an average sequence coverage of ∼26%. HILIC is well suited for the study of hydrophilic PTMs. 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 ∼107–8, the equivalent dynamic range of LC–MS is currently limited to ∼106. 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.