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

• DOI: 10.1038/s41467-022-28879-4
• Publication Date: 2022-03-18T11:12:28Z
• Authors: 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
• Keywords: 0301 basic medicine; Mass spectrometry; Protein Conformation; Science; Q; Serum Albumin, Bovine; Article; Mass Spectrometry; 03 medical and health sciences; Cross-Linking Reagents; Protein folding; Chemical tools; Structural biology; o-Phthalaldehyde; Protein Unfolding

AbstractChemical 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.