Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level
Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level
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DOI：
10.1021/pr7007175
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发表时间：
2008-03-01
影响因子：
4.4
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Hanke, Stefan;Besir, Hueseyin;Mann, Matthias
基于质谱的蛋白质组学可以在单次LC-MS运行中常规地识别数百种蛋白质，并且已经开发了使用稳定同位素在差异处理的样品之间进行相对定量的方法。然而，到目前为止，绝对定量需要在实验工作流程的后期添加标记的标准品，从而由于样品制备而引入变异性。在这里，我们提出了一种新的变体的稳定同位素标记的氨基酸在细胞培养（SILAC）技术称为“绝对SILAC”，允许精确定量的复杂混合物中的选定蛋白质。体内或体外产生的SILAC标记的重组蛋白用作内标物，其直接混合到细胞或组织的裂解物中，这最小化了同位素标记的标准品与其内源性对应物之间的样品处理差异。我们表明，即使在整个细胞裂解物的背景下，也可以在几个数量级上进行定量。我们还设计了一种策略，在混合离子阱仪器上以等于或低于其信噪比的水平对肽进行定量，如此处所示的LTQ-Orbitrap。数据系统触发SILAC标记蛋白质的肽，在窄质量范围内启动离子收集，包括内源性和标记肽。该策略将LTQ-Orbitrap的常规检测限扩展了至少一个数量级，并在LC-MS之前无需任何分级分离即可准确定量细胞裂解物中低至150阿托摩尔的蛋白质。我们使用Absolute SILAC确定HeLa、HepG 2和C2 C12细胞中生长因子受体结合蛋白2（Grb 2）的每个细胞拷贝数为5.5 x 10（5），8.8 × 10（5）和5.7 × 10（5）。
Mass spectrometry based proteomics can routinely identify hundreds of proteins in a single LC-MS run, and methods have been developed for relative quantitation between differentially treated samples using stable isotopes. However, absolute quantitation has so far required addition of a labeled standard late in the experimental workflow, introducing variability due to sample preparation. Here we present a new variant of the stable isotope labeling by amino acids in cell culture (SILAC) technique termed "Absolute SILAC" that allows accurate quantitation of selected proteins in complex mixtures. SILAC-labeled recombinant proteins produced in vivo or in vitro are used as internal standards, which are directly mixed into lysates of cells or tissues, This minimizes differences in sample processing between the isotope-labeled standard and its endogenous counterpart. We show that it is possible to quantify over several orders of magnitude, even in the background of a whole cell lysate. We furthermore devise a strategy to quantify peptides at or below their signal-to-noise level on hybrid ion trap instruments, shown here for the LTQ-Orbitrap. The data system triggers on peptides of the SILAC-labeled protein, initiating ion collection in a narrow mass range including the endogenous and labeled peptide. This strategy extends the regular detection limit of an LTQ-Orbitrap by at least an order of magnitude and accurately quantifies down to 150 attomole of protein in a Cell lysate without any fractionation prior to LC-MS. We use Absolute SILAC to determine the copy number per cell of growth factor receptor-bound protein 2 (Grb2) in HeLa, HepG2, and C2C12 cells to 5.5 x 10(5), 8.8 x 10(5), and 5.7 x 10(5), respectively, in the exponential growth phase.