Stable-isotope dimethyl labeling for quantitative proteomics
Stable-isotope dimethyl labeling for quantitative proteomics
复制标题
DOI：
10.1021/ac0348625
复制
发表时间：
2003-12-15
影响因子：
7.4
通讯作者：
Chen, SH
中科院分区：
文献类型：
--
作者：
Hsu, JL;Huang, SY;Chen, SH
本文报道了一种新的、稳定的同位素标记定量蛋白质组学方法，该方法使用一种简单的试剂甲醛，通过还原胺化作用对Lys的N-末端和E-氨基进行全局标记。Ibis标记策略产生的峰，每个衍生化位点相对于其非衍生化对应物相差28个质量单位，每个衍生化同位素对相差4个质量单位。基于衍生化和非衍生化肽标准品和血红蛋白分子的胰蛋白酶肽的MALDI和LC/ESI-MS/MS光谱分析，该标记反应快速（小于5 min）且完全，没有任何可检测的副产物。产生的a（1）和y（n-1）离子的强度在标记后显著增强，这在大多数非衍生化片段中检测不到。我们进一步测试了基于肽标准品的同位素对和具有已知H/D比的一对定义的蛋白质混合物的分析的方法。使用LC/MS进行定量和LC/MS/MS进行肽测序，结果显示同位素效应可忽略不计，同位素对之间的质量分辨率良好，实验和理论数据之间的相关性良好（误差0-4%）。由同一蛋白质推导的肽段计算的H/D值的相对标准偏差小于13%。定量蛋白质谱的方法的适用性也进行了探讨，通过分析在永生化的E7细胞与砷处理和不处理的核蛋白丰度的变化。
In this paper, we report a novel, stable-isotope labeling strategy for quantitative proteomics that uses a simple reagent, formaldehyde, to globally label the N-terminus and E-amino group of Lys through reductive amination. Ibis labeling strategy produces peaks differing by 28 mass units for each derivatized site relative to its nonderivatized counterpart and 4 mass units for each derivatized isotopic pair. This labeling reaction is fast (less than 5 min) and complete without any detectable byproducts based on the analysis of MALDI and LC/ESI-MS/MS spectra of both derivatized and nonderivatized peptide standards and tryptic peptides of hemoglobin molecules. The intensity of the a(1) and y(n-1) ions produced, which were not detectable from most of the nonderivatized fragments, was substantially enhanced upon labeling. We further tested the method based on the analysis of an isotopic pair of peptide standards and a pair of defined protein mixtures with known H/D ratios. Using LC/MS for quantification and LC/MS/MS for peptide sequencing, the results show a negligible isotopic effect, a good mass resolution between the isotopic pair, and a good correlation between the experimental and theoretical data (errors 0-4%). The relative standard deviation of H/D values calculated from peptides deduced from the same protein are less than 13%. The applicability of the method for quantitative protein profiling was also explored by analyzing changes in nuclear protein abundance in an immortalized E7 cell with and without arsenic treatment.