Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics
Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics
复制标题
DOI：
10.1038/nprot.2010.192
复制
发表时间：
2011-02-01
期刊：
影响因子：
14.8
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Geiger, Tamar;Wisniewski, Jacek R.;Mann, Matthias
基于质谱（MS）的蛋白质组学越来越多地应用于定量格式，通常基于用化学或代谢引入的稳定同位素标记样品。在细胞培养中氨基酸稳定同位素标记（SILAC）方法中，两个细胞群在存在重或轻氨基酸（通常是赖氨酸和/或精氨酸）的情况下培养，其中一个细胞群受到扰动，然后将两者组合在一起处理。在本研究中，我们描述了一种不同的方法-使用SILAC作为内部或“峰值”标准-其中SILAC仅用于产生重标记参考蛋白或蛋白质组。这些是在细胞裂解后和蛋白质消化前添加到所研究的蛋白质组中。因此，实际的实验与标注过程完全分离。Spike-in SILAC非常经济，稳健，原则上适用于所有基于细胞或组织的蛋白质组学分析。应用范围从单个蛋白质的绝对定量到整个蛋白质组的定量。在分析整个组织或生物体的蛋白质组时，Spike-in SILAC特别有利。该方案描述了与超级silac峰值相关的组织样本的定量分析，这是一种由五种silac标记的细胞系组成的混合物，可以准确地代表组织。它包括峰入式SILAC标准品的选择和制备，样品制备程序，以及结果的分析和评价。
Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format, often based on labeling of samples with stable isotopes that are introduced chemically or metabolically. In the stable isotope labeling by amino acids in cell culture (SILAC) method, two cell populations are cultured in the presence of heavy or light amino acids (typically lysine and/or arginine), one of them is subjected to a perturbation, and then both are combined and processed together. In this study, we describe a different approach-the use of SILAC as an internal or 'spike-in' standard-wherein SILAC is only used to produce heavy labeled reference proteins or proteomes. These are added to the proteomes under investigation after cell lysis and before protein digestion. The actual experiment is therefore completely decoupled from the labeling procedure. Spike-in SILAC is very economical, robust and in principle applicable to all cell-or tissue-based proteomic analyses. Applications range from absolute quantification of single proteins to the quantification of whole proteomes. Spike-in SILAC is especially advantageous when analyzing the proteomes of whole tissues or organisms. The protocol describes the quantitative analysis of a tissue sample relative to super-SILAC spike-in, a mixture of five SILAC-labeled cell lines that accurately represents the tissue. It includes the selection and preparation of the spike-in SILAC standard, the sample preparation procedure, and analysis and evaluation of the results.