Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC).
Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC).
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DOI：
10.1016/j.jprot.2012.04.035
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发表时间：
2012-06-27
影响因子：
3.3
通讯作者：
Maudsley S
中科院分区：
文献类型：
--
作者：
Park SS;Wu WW;Zhou Y;Shen RF;Martin B;Maudsley S
通过引入细胞培养中氨基酸的稳定同位素标记（SILAC），结合高分辨率质谱，已经大大促进了细胞培养中准确和可靠的定量蛋白质组学。然而，SILAC技术通常存在几个主要的定量误差来源，即同位素氨基酸的不完全掺入、甘氨酸至脯氨酸的转化以及最终样品混合中的实验误差。数据集规范化是对此类错误的广泛采用的解决方案，然而这可能无法完全防止引入不正确的表达比率。在这里，我们证明了SILAC实验的标签交换复制能够有效地纠正实验误差，通过使用定量蛋白质组学和磷酸化蛋白质组学的配体治疗神经细胞培养物的平均比率在单个重复测量。此外，这种策略被成功地应用于SILAC三联体实验，这提出了一个更复杂的实验矩阵，受不完全标记和脯氨酸到脯氨酸的转换。基于我们的研究结果，我们建议SILAC实验的设计应纳入标签交换重复，以提高表达率的可靠性。
Accurate and reliable quantitative proteomics in cell culture has been considerably facilitated by the introduction of the stable isotope labeling by amino acids in cell culture (SILAC), combined with high resolution mass spectrometry. There are however several major sources of quantification errors that commonly occur with SILAC techniques, i.e. incomplete incorporation of isotopic amino acids, arginine-to-proline conversion, and experimental errors in final sample mixing. Dataset normalization is a widely adopted solution to such errors, however this may not completely prevent introducing incorrect expression ratios. Here we demonstrate that a label-swap replication of SILAC experiments was able to effectively correct experimental errors by averaging ratios measured in individual replicates using quantitative proteomics and phosphoproteomics of ligand treatment of neural cell cultures. Furthermore, this strategy was successfully applied to a SILAC triplet experiment, which presents a much more complicated experimental matrix, affected by both incomplete labeling and arginine-to-proline conversion. Based on our results, we suggest that SILAC experiments should be designed to incorporate label-swap replications for enhanced reliability in expression ratios.
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代谢途径分析的生物信息学方法。
DOI：
10.1007/978-1-61779-160-4_5
发表时间：
2011
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Maudsley S;Chadwick W;Wang L;Zhou Y;Martin B;Park SS
通讯作者：
Park SS
影响因子：
4.4
作者：
Cox, Juergen;Neuhauser, Nadin;Mann, Matthias
通讯作者：
Mann, Matthias
防止胚胎干细胞 SILAC 实验中的氨基酸转换
影响因子：
7
作者：
Bendall, Sean C.;Hughes, Chris;Lajoie, Gilles A.
通讯作者：
Lajoie, Gilles A.
影响因子：
7
作者：
Ross, PL;Huang, YLN;Pappin, DJ
通讯作者：
Pappin, DJ
DOI：
10.1074/mcp.m110.000208
发表时间：
2010-07
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Bicho CC;de Lima Alves F;Chen ZA;Rappsilber J;Sawin KE
通讯作者：
Sawin KE