Amino acid residue specific stable isotope labeling for quantitative proteomics
Amino acid residue specific stable isotope labeling for quantitative proteomics
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DOI：
10.1002/rcm.831
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发表时间：
2002-01-01
影响因子：
2
通讯作者：
Chen, X
中科院分区：
文献类型：
--
作者：
Zhu, HN;Pan, SQ;Chen, X
近年来，各种稳定同位素标记(SIL)技术不断涌现，以提高蛋白质质谱学定量分析的效率和准确性。我们开发了一种质量标记策略，在细胞生长过程中以特定残基的方式将稳定同位素标记的氨基酸整合到细胞蛋白质中。在这项研究中，我们进一步扩展了这种特定残基的sIL方法，以准确地定量不同细胞群体中的蛋白质丰度。对于在正常和标记培养液中生长的细胞中表达水平相同的蛋白质，正常(轻)和标记(重)同位素峰的相对面积与细胞混合比率线性相关。这种方法首次被用来确定锌反应转录因子Zap1对酵母蛋白质组的影响。从PAGE凝胶中随机选择10个蛋白质点，通过同位素比率很容易地确定它们在野生型和zap1Delta细胞中的差异蛋白质表达水平。在zap1Delta突变株中，蛋氨酸合成酶(Met6)的表达上调了4倍以上，而其他9种蛋白的表达水平保持不变。此外，我们应用这一策略来研究人皮肤成纤维细胞对辐射的细胞反应。对随机选择的一条蛋白质条带进行分析，发现一个新蛋白的表达水平随着辐射的增加而增加两倍，而对照蛋白的表达水平保持不变。这一策略通常适用于使用任何特定类型的氨基酸作为标记前体来准确地定量蛋白质的相对丰度。2002年由John Wiley Sons，Ltd.出版。
Various stable isotope labeling (SIL) techniques have recently emerged to improve the efficiency and accuracy of protein quantitation by mass spectrometry (MS). We have developed a mass-tagging strategy to incorporate stable isotope tagged amino acids into cellular proteins in a residue-specific manner during cell growth. In this study, we further extend this residue-specific SIL approach to the accurate quantitation of protein abundances in different cell populations. For proteins whose expression levels are the same in cells grown in the normal and labeled media, the relative areas of the normal (light) and labeled (heavy) isotopic peaks are linearly correlated with the cells mixing ratios. This approach was first used to determine the effect of the zinc-responsive transcription factor Zap1 on the yeast proteome. Ten protein spots from a PAGE gel were chosen randomly and their differential protein expression levels in wild-type and zap1Delta cells were readily determined by the isotopic ratio. Methionine synthase (Met6) was identified to be up-regulated more than four times in the zap1Delta mutant strain whereas the expression level of other nine proteins remained unchanged. Further, we applied this strategy to study the cellular response to radiation in human skin fibroblast cells. Analyzing one protein band randomly selected from SDS-PAGE, the expression level of a novel protein was found to increase two-fold in response to radiation whereas the expression level of a control protein remained unchanged. This strategy is generally applicable using any particular type of amino acid as the labeling precursors for accurate quantitation of protein relative abundances. Published in 2002 by John Wiley Sons, Ltd.