Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics.
Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics.
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DOI：
10.1021/pr500630a
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发表时间：
2014-09-05
影响因子：
4.4
通讯作者：
Ong, Shao-En
中科院分区：
文献类型：
--
作者：
Lau, Ho-Tak;Suh, Hyong Won;Golkowski, Martin;Ong, Shao-En
稳定同位素标记在基于质谱学的蛋白质组学中被广泛用于蛋白质的编码和定量。我们比较了代谢标记和细胞培养中氨基酸稳定同位素标记(SILAC)和稳定同位素二甲基标记化学标记，发现它们在蛋白质组分析和亲和下拉实验中具有相当的准确性和定量动态范围。在单液相色谱质谱分析中同时分析SILAC和二甲基标记的样品，可以在分析条件下最大限度地减少差异，从而可以比较样品处理过程中引入的定量误差。我们发现SILAC比二甲基标记法具有更好的重复性。由于来自代谢性标记群体的蛋白质可以在蛋白水解性消化之前结合在一起，SILAC特别适合进行大量样品处理的研究，例如通过翻译后修饰对多肽进行分级和浓缩。我们在下拉实验中比较了这两种方法，使用了一种激酶抑制剂达沙替尼和标记的Grb2-SH2蛋白作为亲和诱饵。我们描述了一种应用于溶液内和凝胶内蛋白质消化的StageTip二甲基标记法。比较消化后同位素标记对定量准确性的影响，我们演示了特定的实验设计如何从SILAC等代谢标记方法中获得最大好处，以及通过稳定同位素-二甲基标记进行化学标记可以是一种实用的替代方案。
Stable isotope labeling is widely used to encode and quantify proteins in mass-spectrometry-based proteomics. We compared metabolic labeling with stable isotope labeling by amino acids in cell culture (SILAC) and chemical labeling by stable isotope dimethyl labeling and find that they have comparable accuracy and quantitative dynamic range in unfractionated proteome analyses and affinity pull-down experiments. Analyzing SILAC- and dimethyl-labeled samples together in single liquid chromatography–mass spectrometric analyses minimizes differences under analytical conditions, allowing comparisons of quantitative errors introduced during sample processing. We find that SILAC is more reproducible than dimethyl labeling. Because proteins from metabolically labeled populations can be combined before proteolytic digestion, SILAC is particularly suited to studies with extensive sample processing, such as fractionation and enrichment of peptides with post-translational modifications. We compared both methods in pull-down experiments using a kinase inhibitor, dasatinib, and tagged GRB2-SH2 protein as affinity baits. We describe a StageTip dimethyl-labeling protocol that we applied to in-solution and in-gel protein digests. Comparing the impact of post-digest isotopic labeling on quantitative accuracy, we demonstrate how specific experimental designs can benefit most from metabolic labeling approaches like SILAC and situations where chemical labeling by stable isotope-dimethyl labeling can be a practical alternative.
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影响因子：
48
作者：
Hebert, Alexander S.;Merrill, Anna E.;Bailey, Derek J.;Still, Amelia J.;Westphall, Michael S.;Strieter, Eric R.;Pagliarini, David J.;Coon, Joshua J.
通讯作者：
Coon, Joshua J.
影响因子：
3.4
作者：
Asara, John M.;Christofk, Heather R.;Cantley, Lewis C.
通讯作者：
Cantley, Lewis C.
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作者：
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通讯作者：
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影响因子：
4.4
作者：
Cox, Juergen;Neuhauser, Nadin;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
3.4
作者：
GUY, HR
通讯作者：
GUY, HR