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; Xiaoguang Lei

doi:10.1038/s41467-022-28879-4

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

Nat Commun. 2022 Mar 18;13(1):1468. doi: 10.1038/s41467-022-28879-4.

Authors

Jian-Hua Wang^#^{1

2}, Yu-Liang Tang^#³, Zhou Gong⁴, Rohit Jain⁵, Fan Xiao³, Yu Zhou^{1

2}, Dan Tan¹, Qiang Li³, Niu Huang^{1

2}, Shu-Qun Liu⁶, Keqiong Ye^{7

8}, Chun Tang^{9

10}, Meng-Qiu Dong^{11

12}, Xiaoguang Lei¹³

Affiliations

¹ National Institute of Biological Sciences (NIBS), 102206, Beijing, China.
² Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, 102206, Beijing, China.
³ Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Science, Peking University, 100871, Beijing, China.
⁴ Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, 430071, Wuhan, Hubei, China.
⁵ University of Massachusetts Medical School, Worcester, 01605, USA.
⁶ State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, 650091, Kunming, Yunnan, China.
⁷ Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101, Beijing, China.
⁸ University of Chinese Academy of Sciences, 100049, Beijing, China.
⁹ Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Science, Peking University, 100871, Beijing, China. Tang_Chun@pku.edu.cn.
¹⁰ Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, 430071, Wuhan, Hubei, China. Tang_Chun@pku.edu.cn.
¹¹ National Institute of Biological Sciences (NIBS), 102206, Beijing, China. dongmengqiu@nibs.ac.cn.
¹² Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, 102206, Beijing, China. dongmengqiu@nibs.ac.cn.
¹³ Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Science, Peking University, 100871, Beijing, China. xglei@pku.edu.cn.

^# Contributed equally.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cross-Linking Reagents / chemistry
Mass Spectrometry / methods
Protein Conformation
Protein Unfolding*
Serum Albumin, Bovine / chemistry
o-Phthalaldehyde*

Substances

Cross-Linking Reagents
Serum Albumin, Bovine
o-Phthalaldehyde