Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
J Proteomics. 2013 Aug 2;88:14-26. doi: 10.1016/j.jprot.2012.10.009. Epub 2012 Oct 22.
Several quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or chemical isotope labeling with MS based quantification or chemical labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale quantitative proteomics experiments, comparing SILAC, dimethyl and TMT labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and chemical labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used.
近年来，出现了几种基于定量质谱的技术来探究蛋白质组的复杂性、相互连接性和动态性质。目前，最流行的方法是使用代谢或化学同位素标记结合基于 MS 的定量，或使用等重同位素标记结合基于 MS/MS 的定量进行化学标记。在这里，我们通过系统的独立大规模定量蛋白质组学实验来评估三种最流行方法的性能，比较 SILAC、二甲基和 TMT 标记策略。尽管这三种方法都有其优点和缺点，但我们的数据表明，使用经典（基于 MS2 的）shotgun 方法，所有三种方法都可以达到相似数量的鉴定蛋白深度。仅使用 MS2 的 TMT 定量会受到共分离的严重影响，导致精密度和准确度受损。通过使用基于 MS3 的采集可以部分解决这个问题，但代价是显著减少了定量的蛋白数量。有趣的是，SILAC 和基于 MS 的化学标记产生的结果几乎相同，与使用的数据库搜索算法无关。
