Comparison of stable-isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression
Comparison of stable-isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression
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DOI：
10.1002/rcm.5151
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发表时间：
2011-09-15
影响因子：
2
通讯作者：
Muddiman, David C.
中科院分区：
文献类型：
--
作者：
Collier, Timothy S.;Randall, Shan M.;Muddiman, David C.
蛋白质定量是基于质谱 (MS) 的蛋白质组学的主要目标之一，并且存在许多策略来实现它。有几种方法涉及使用化学衍生化、O-18 酶催化掺入或细胞或组织培养物中的代谢标记掺入稳定同位素标记。这些技术可能成本或时间过高或不适合感兴趣的生物系统。无标记技术，包括利用集成离子丰度和光谱计数的技术，提供了基于稳定同位素的方法的替代方法。在此，我们比较了细胞培养物中氨基酸的稳定同位素标记 (SILAC) 与光谱计数，以对人类胚胎干细胞在三个时间点分化为滋养外胚层进行定量。我们的光谱计数实验策略在三个时间点鉴定了 2641 个蛋白质组，平均序列覆盖率为 30.3%，其中 1837 个蛋白质组可以通过超过 5 个光谱计数进行定量。 SILAC 定量能够识别 1369 个蛋白质组，平均覆盖率为 24.7%，其中 1027 个蛋白质组可以在所有时间点进行定量。在此背景下，我们进一步探讨了每种策略的蛋白质组覆盖能力、定量变化以及每种技术对检测相对蛋白质表达变化的相对敏感性。版权所有 (C) 2011 John Wiley & Sons, Ltd.
Protein quantification is one of the principal goals of mass spectrometry (MS)-based proteomics, and many strategies exist to achieve it. Several approaches involve the incorporation of a stable-isotope label using either chemical derivatization, enzymatically catalyzed incorporation of O-18, or metabolic labeling in a cell or tissue culture. These techniques can be cost or time prohibitive or not amenable to the biological system of interest. Label-free techniques including those utilizing integrated ion abundance and spectral counting offer an alternative to stable-isotope-based methodologies. Herein, we present the comparison of stable-isotope labeling of amino acids in cell culture (SILAC) with spectral counting for the quantification of human embryonic stem cells as they differentiate toward the trophectoderm at three time points. Our spectral counting experimental strategy resulted in the identification of 2641 protein groups across three time points with an average sequence coverage of 30.3%, of which 1837 could be quantified with more than five spectral counts. SILAC quantification was able to identify 1369 protein groups with an average coverage of 24.7%, of which 1027 could be quantified across all time points. Within this context we further explore the capacity of each strategy for proteome coverage, variation in quantification, and the relative sensitivity of each technique to the detection of change in relative protein expression. Copyright (C) 2011 John Wiley & Sons, Ltd.