A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen
A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen
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DOI：
10.1002/pmic.200700426
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发表时间：
2008-03-01
期刊：
影响因子：
3.4
通讯作者：
Cantley, Lewis C.
中科院分区：
文献类型：
--
作者：
Asara, John M.;Christofk, Heather R.;Cantley, Lewis C.
为了评估蛋白质的生物学功能及其修改，以了解细胞内的信号传导机制以及对疾病的特定生物标志物，重要的是在不同的实验条件下获得定量信息很重要。稳定的同位素标记是一种强大的方法，用于准确确定蛋白质和PTM的水平的变化；但是，使用大多数商业质谱仪，同位素标记实验的动态范围有限，导致信号变化比小于20：1。诸如光谱计数之类的蛋白质组学相对定量的无标签方法已经越来越受欢迎，因为不需要额外的化学成分。在这里，我们显示了一种基于从数据依赖性运行中收集的肽MS/MS光谱的TIC相对定量的无标签方法，可以有效地用作定量度量，并扩展了在同位素标记实验上的动态范围，从在与磷酸酪氨酸残基结合的蛋白质中类似于60：1。
In order to assess the biological function of proteins and their modifications for understanding signaling mechanisms within cells as well as specific biomarkers to disease, it is important that quantitative information be obtained under different experimental conditions. Stable isotope labeling is a powerful method for accurately determining changes in the levels of proteins and PTMs; however, isotope labeling experiments suffer from limited dynamic range resulting in signal change ratios of less than similar to 20:1 using most commercial mass spectrometers. Label-free approaches to relative quantification in proteomics such as spectral counting have gained popularity since no additional chemistries are needed. Here, we show a label-free method for relative quantification based on the TIC from peptide MS/MS spectra collected from data-dependent runs can be used effectively as a quantitative measure and expands the dynamic range over isotope labeling experiments allowing for abundance differences up to similar to 60:1 in a screen for proteins that bind to phosphotyrosine residues.