Quantitative proteome and phosphoproteome analyses of cultured cells based on SILAC labeling without requirement of serum dialysis
Quantitative proteome and phosphoproteome analyses of cultured cells based on SILAC labeling without requirement of serum dialysis
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DOI：
10.1039/b921379a
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发表时间：
2010-01-01
影响因子：
--
通讯作者：
Ishihama, Yasushi
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文献类型：
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作者：
Imami, Koshi;Sugiyama, Naoyuki;Ishihama, Yasushi
透析血清的使用对于应用细胞培养中氨基酸的常规稳定同位素标记 (SILAC) 方法至关重要，以实现定量蛋白质组学中蛋白质的完全标记。在这里，我们首先评估了透析血清对激素敏感乳腺癌MCF-7细胞蛋白质组和磷酸蛋白质组的影响，发现透析血清通过激素受体影响信号系统相关蛋白的表达，与使用非透析血清相比，引起磷酸蛋白质组的显着变化。我们还评估了其他 9 种细胞系，包括 HeLa、HEK293 和 Panc1，发现血清透析对蛋白质组和磷酸蛋白质组表达谱的影响因细胞类型而异。为了避免这些问题，我们建立了一种基于 SILAC 的定量方法，无需血清透析。我们的简单方法是基于使用非透析血清用两种不同质量的重氨基酸对两个细胞群进行双重标记。使用我们的 SILAC 方法和非透析血清，我们成功定量了拉帕替尼（一种 EGFR1/Her2 双激酶抑制剂）诱导的 MCF-7 细胞的磷酸化蛋白质组。由于采用双重标记方法，我们的方法广泛适用于由于任何原因（例如，蛋白质标记）不完整的培养细胞。例如，由于使用非透析血清或低生长率。
The use of dialyzed serum is essential in the application of the conventional stable isotope labeling by amino acids in cell culture (SILAC) approach to achieve complete labeling of proteins for quantitative proteomics. Here, we first evaluated the impact of dialyzed serum on the proteome and phosphoproteome of hormone-sensitive breast cancer MCF-7 cells and found that dialyzed serum influenced the expression of proteins related to signaling systems via hormone receptors, inducing a marked change of the phosphoproteome compared with the use of non-dialyzed serum. We also evaluated 9 other cell lines, including HeLa, HEK293 and Panc1, and found that the influence of serum dialysis on the expression profiles of the proteome and phosphoproteome varied, depending on the cell type. To avoid these problems, we established a SILAC-based quantification approach without the requirement of serum dialysis. Our simple approach is based on dual labeling of two populations of cells with two kinds of heavy amino acids of different mass, using non-dialyzed serum. Using our SILAC approach with non-dialyzed serum, we successfully quantified the phosphoproteome of MCF-7 cells induced by lapatinib, an EGFR1/Her2 dual kinase inhibitor. Because of the dual labeling approach, our method is widely applicable to cultured cells in which protein labeling is incomplete for any reason, e. g., owing to the use of non-dialyzed serum or a low growth rate.