Characterization of protein unfolding by fast cross-linking mass spectrometry using di-ortho-phthalaldehyde cross-linkers

Jian-Hua Wang, Yu-Liang Tang, Zhou Gong, Rohit Jain, Fan Xiao, Yu Zhou, Dan Tan, Qiang Li, Niu Huang, Shu-Qun Liu, Keqiong Ye, Chun Tang (), Meng-Qiu Dong () and Xiaoguang Lei ()
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Jian-Hua Wang: National Institute of Biological Sciences (NIBS)
Yu-Liang Tang: Peking University
Zhou Gong: Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences
Rohit Jain: University of Massachusetts Medical School
Fan Xiao: Peking University
Yu Zhou: National Institute of Biological Sciences (NIBS)
Dan Tan: National Institute of Biological Sciences (NIBS)
Qiang Li: Peking University
Niu Huang: National Institute of Biological Sciences (NIBS)
Shu-Qun Liu: Yunnan University
Keqiong Ye: Chinese Academy of Sciences
Chun Tang: Peking University
Meng-Qiu Dong: National Institute of Biological Sciences (NIBS)
Xiaoguang Lei: Peking University

Nature Communications, 2022, vol. 13, issue 1, 1-16

Abstract: Abstract Chemical cross-linking of proteins coupled with mass spectrometry is widely used in protein structural analysis. In this study we develop a class of non-hydrolyzable amine-selective di-ortho-phthalaldehyde (DOPA) cross-linkers, one of which is called DOPA2. Cross-linking of proteins with DOPA2 is 60–120 times faster than that with the N-hydroxysuccinimide ester cross-linker DSS. Compared with DSS cross-links, DOPA2 cross-links show better agreement with the crystal structures of tested proteins. More importantly, DOPA2 has unique advantages when working at low pH, low temperature, or in the presence of denaturants. Using staphylococcal nuclease, bovine serum albumin, and bovine pancreatic ribonuclease A, we demonstrate that DOPA2 cross-linking provides abundant spatial information about the conformations of progressively denatured forms of these proteins. Furthermore, DOPA2 cross-linking allows time-course analysis of protein conformational changes during denaturant-induced unfolding.

Date: 2022
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DOI: 10.1038/s41467-022-28879-4

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