CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Proteomics. 2012 Nov;12(21):3129-37. doi: 10.1002/pmic.201200162. Epub 2012 Oct 1.
Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post-digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two-fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 10(5) Hela cells).
近年来，已经开发出了各种酶反应器和在线酶消化策略。这些反应器大大提高了定性蛋白质组学中的检测灵敏度和蛋白质组覆盖率。然而，这些设备在蛋白质消化过程中存在更高的错误切割率。因此，我们研究了在线酶消化对使用化学或代谢同位素标记方法进行定量蛋白质组学定量准确性的影响。当样品在不同等分试样中进行消化和化学同位素标记时，不完全消化会在比较定量中引入一些意外的变化。即使使用在线酶消化后进行化学同位素标记，并将比值的 CV 控制在重复分析中小于 50%，相同蛋白质等分试样的处理也会导致约 10%的定量蛋白的比值大于两倍，而理论上该比值应为 1:1。有趣的是，当处理代谢同位素标记的样品时，酶反应器中的不完全消化不是问题，因为在反应器中同时消化之前，蛋白质已经在体内进行了同位素标记。我们的结果还表明，即使使用有限数量的代谢同位素标记样品（从 10^5 个 HeLa 细胞中比较定量了 1683 种蛋白质），在线酶消化也可以实现定量蛋白质组学比较中的高定量准确性和高蛋白质组覆盖率。
