Department of Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany.
Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany.
Anal Chem. 2020 Mar 3;92(5):4016-4022. doi: 10.1021/acs.analchem.9b05559. Epub 2020 Feb 18.
Proteome-wide cross-linking studies have spurred great interest as they facilitate structural probing of protein interactions in living cells and organisms. However, current studies have a bias for high-abundant proteins. In this study we demonstrate both experimentally and by a kinetic model that this bias is also caused by the propensity of cross-links to preferentially form on high abundant proteins and not by the inability to detect cross-links due to limitations in current technology. We further show, by using both an in vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that parameters optimized toward a pseudo first order kinetics model result in a significant increase in the detection of lower-abundant proteins on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide cross-linking studies and demonstrates how to address a larger part of the proteome by cross-linking.
蛋白质组范围的交联研究引起了极大的兴趣，因为它们促进了在活细胞和生物体中对蛋白质相互作用的结构探测。然而，目前的研究偏向于高丰度蛋白质。在这项研究中，我们通过实验和动力学模型证明，这种偏差也是由于交联倾向于优先在高丰度蛋白质上形成，而不是由于当前技术的限制而无法检测到交联。我们进一步通过使用细胞拥挤环境的体外模拟物和真核细胞裂解物表明，针对拟一级动力学模型进行优化的参数可显著增加在蛋白质组范围内检测低丰度蛋白质的数量。因此，我们的研究解释了当前蛋白质组范围交联研究中的一个主要限制的原因，并展示了如何通过交联来解决蛋白质组的更大部分。
