A genetic engineering solution to the "arginine conversion problem" in stable isotope labeling by amino acids in cell culture (SILAC).
A genetic engineering solution to the "arginine conversion problem" in stable isotope labeling by amino acids in cell culture (SILAC).
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DOI：
10.1074/mcp.m110.000208
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发表时间：
2010-07
期刊：
影响因子：
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通讯作者：
Sawin KE
中科院分区：
文献类型：
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作者：
Bicho CC;de Lima Alves F;Chen ZA;Rappsilber J;Sawin KE
细胞培养物中氨基酸的稳定同位素标记 (SILAC) 为蛋白质组学定量提供了一种简单的工具。然而，与 SILAC 相关的一个问题是标记的精氨酸在体内转化为其他氨基酸（通常是脯氨酸）。我们发现裂殖酵母粟酒裂殖酵母中的精氨酸转化水平极高，因此用重精氨酸标记细胞会导致标签不希望地掺入基本上所有脯氨酸池以及大部分谷氨酸、谷氨酰胺和赖氨酸池中。我们发现，可以通过使用高度稳健且易于实施的方法删除涉及精氨酸分解代谢的基因来防止这种情况。删除裂殖酵母精氨酸酶基因或单个鸟氨酸转氨酶基因，再加上对生长培养基进行小修改以改善突变菌株中精氨酸的吸收，足以消除基本上所有的精氨酸转化。我们证明了我们的方法在细胞分裂前后蛋白质的大规模定量分析中的有用性；上调和下调的蛋白质，包括一种参与分隔的新蛋白质，均被成功鉴定。这种解决“精氨酸转化问题”的策略可能更广泛地适用于适合基因操作的生物体。
Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the “arginine conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.