SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function
SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function
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DOI：
10.1016/j.cell.2008.05.033
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发表时间：
2008-07-25
期刊：
影响因子：
64.5
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Krueger, Marcus;Moser, Markus;Mann, Matthias
细胞培养中氨基酸稳定同位素标记技术（SILAC）已成为基于质谱（MS）的蛋白质组学定量分析的通用工具。在这里，我们用含有天然赖氨酸或（13）C（6）取代赖氨酸的饮食完全标记小鼠。小鼠被标记了四代重饮食，发育，生长和行为没有受到影响。掺入水平的MS分析允许测定来自血细胞和器官的蛋白质的掺入率。F2代在所有检测器官中完全标记。来自缺乏β 1整联蛋白、β-Parvin或整联蛋白尾结合蛋白Kindlin-3表达的各种器官的SILAC分析证实了它们的缺乏，并揭示了Kindlin-3缺陷型红细胞中红细胞膜骨架的结构缺陷。SILAC-小鼠方法是一种多功能工具，通过该方法可以定量比较基因敲除小鼠的蛋白质组，从而确定复杂体内条件下的蛋白质功能。
Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of beta 1 integrin, beta-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions.