Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates
Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates
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DOI：
10.1002/pmic.200800297
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发表时间：
2008-11-01
期刊：
影响因子：
3.4
通讯作者：
Mohammed, Shabaz
中科院分区：
文献类型：
--
作者：
Boersema, Paul J.;Aye, Thin Thin;Mohammed, Shabaz
稳定同位素标记是目前定量蛋白质组学中最强大的方法之一。使用代谢或化学标记在蛋白质和肽水平上进行稳定同位素标记。在这里，我们提出了一种简单且经济高效的三重定量方法，该方法基于肽水平的稳定同位素二甲基标记。本文中，所有蛋白水解肽均在其α-和ε-氨基处进行化学标记。我们使用甲醛的三种不同同位素异构体来平行分析三种不同的样品。这些标签在生成的肽三联体中的峰之间提供了至少 4 Da 的质量差异。使用典型的“鸟枪”蛋白质组学实验，基于细胞裂解物的定量分析对该方法进行评估。虽然通过引入三个标记增加了肽的复杂性，但使用 60 μg 起始材料仍然可以鉴定超过 1300 种蛋白质，其中每种蛋白质至少使用 4 个肽可以定量超过 600 种蛋白质。在使用固定化 cAMP 的化学蛋白质组学实验中，三链体标记进一步用于区分特异性和非特异性 cAMP 结合蛋白。因此，可以量化两个以上数量级的丰度比差异。
Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their alpha- and epsilon-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 mu g of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.