CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, China.
University of Chinese Academy of Sciences, Beijing 100039, China.
Anal Chem. 2022 May 31;94(21):7551-7558. doi: 10.1021/acs.analchem.2c00335. Epub 2022 May 16.
Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference. Followed by a two-step enrichment method based on click chemistry at the peptide level, 13,098 crosslinked peptides (5068 inter-crosslinked peptides and 8030 intra-crosslinked peptides) were identified under the data threshold of peptide-spectrum matches (PSMs) ≥2 on the basic of the FDR control of 1%, which was the most comprehensive dataset for homo species cells by a non-cleavable crosslinker. Besides, the interactome network comprising 1519 proteins connected by 2913 interaction edges in various intracellular compartments, as well as 80S ribosome structural dynamics, were characterized, showing the great potential of our crosslinking approach in minutes. All these results demonstrated that our developed BSP could provide a valuable toolkit for the in-depth analysis of protein-protein interactions (PPIs) and protein architectures with finer temporal resolution.
化学交联结合质谱（CXMS）已成为获取蛋白质复合物动态构象和相互作用界面的强大技术。由于交联剂的细胞膜通透性差、化学反应性和生物相容性有限，交联以更高的时间分辨率和更高的覆盖度来捕获蛋白质复合物的动力学是有吸引力的，但具有挑战性。在这项工作中，开发了一种带有炔基标记的三功能交联剂双（琥珀酰亚胺基）（BSP），它涉及紧凑的尺寸、适当的两亲性和富集能力，可提高细胞膜通透性，并在 5 分钟内实现有效的交联，而不会对细胞造成明显干扰。随后，通过基于肽水平的点击化学的两步富集方法，在肽谱匹配（PSM）≥2 的数据阈值下，鉴定了 13098 个交联肽（5068 个互交联肽和 8030 个内交联肽），这是使用不可裂解交联剂对同种细胞进行的最全面的数据集。此外，还对包含 1519 个蛋白质的互作网络进行了表征，这些蛋白质通过 2913 个在各种细胞内隔室中的相互作用边缘连接，以及 80S 核糖体结构动力学，表明我们的交联方法在数分钟内具有很大的潜力。所有这些结果表明，我们开发的 BSP 可以为更深入地分析蛋白质-蛋白质相互作用（PPIs）和蛋白质结构提供有价值的工具包，具有更精细的时间分辨率。
