Prevention of amino acid conversion in SILAC experiments with embryonic stem cells
Prevention of amino acid conversion in SILAC experiments with embryonic stem cells
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防止胚胎干细胞 SILAC 实验中的氨基酸转换
DOI：
10.1074/mcp.m800113-mcp200
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发表时间：
2008-09-01
影响因子：
7
通讯作者：
Lajoie, Gilles A.
中科院分区：
文献类型：
--
作者：
Bendall, Sean C.;Hughes, Chris;Lajoie, Gilles A.
最近在定量蛋白质组学中使用培养中氨基酸的稳定同位素标记(SILAC)的研究提到了在细胞中由同位素编码的精氨酸转化为脯氨酸的问题。所得到的转换后的脯氨酸肽将重肽离子信号分开，导致与轻肽离子信号相比不准确。这一点特别值得关注，因为在蛋白质组实验中，它可以影响多达一半的多肽。补偿和限制无意转换的策略已经得到证明，但没有一种策略被证明可以防止这种情况。此外，这些方法与SILAC标记相结合，在大规模应用于敏感细胞类型包括来自小鼠和人类的胚胎干细胞(ESCs)时被证明是有问题的。在这里，我们表明，在SILAC介质中提供低至200毫克/升的L-脯氨酸，精氨酸到脯氨酸的转化可以完全检测不到。与此同时，在用同位素编码的精氨酸进行标记方面没有妥协，这表明没有观察到从Pro补充剂的反向转化。因此，当补充了脯氨酸时，即使在最糟糕的转化情况下，也可以正确地解释“轻”和“重”的多肽比例。通过将这些原则扩展到ESC培养方案和试剂，我们能够在没有饲养细胞的情况下常规地标记小鼠和人类ESCs，而不会影响多能性表型。这项研究提供了一种最简单的方案，以防止精氨酸标记SILAC实验中的脯氨酸伪影。此外，它还提出了一种健壮的、无饲养层细胞的方案，用于对来自小鼠和人类的ESCs进行SILAC实验。
Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotope-coded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter L-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of "light" and "heavy" peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human.